in vitro interactions between pseudorabies virus and

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... National Institute of. Allergy and Infectious Diseases (to R.J.E. and G.H.C. respectively). ...... 31st International Herpesvirus Workshop, Seattle, Washington, VS.
LABORATORIUM VOOR VIROLOGIE VAKGROEP VIROLOGIE, PARASITOLOGIE EN IMMUNOLOGIE FACULTEIT DIERGENEESKUNDE

IN VITRO INTERACTIONS BETWEEN PSEUDORABIES VIRUS AND TRIGEMINAL GANGLION NEURONS WITH IMPLICATIONS FOR VIRUS SPREAD AND LATENCY

Nick De Regge

Proefschrift voorgedragen tot het behalen van de graad van Doctor in de Diergeneeskundige Wetenschappen, 2007

Promotoren: Prof. Dr. H.W. Favoreel Prof. Dr. H.J. Nauwynck

TABLE OF CONTENTS List of abbreviations CHAPTER 1: INTRODUCTION..........................................................................................1 1.1. PSEUDORABIES VIRUS, AN ALPHAHERPESVIRUS .............................................................2 1.1.1. Classification and introduction .............................................................................2 1.1.2. Virion structure.....................................................................................................3 1.2. TRIGEMINAL GANGLION NEURONS .................................................................................4 1.2.1. Anatomical structure.............................................................................................4 1.2.2. Primary target cells for different alphaherpesviruses ............................................5 1.3. PRODUCTIVE ALPHAHERPESVIRUS INFECTION IN NEURONS AND SUBSEQUENT VIRAL SPREAD ...............................................................................................................................6 1.3.1. Entry.....................................................................................................................6 1.3.2. Transport to the nucleus........................................................................................8 1.3.3. Intranuclear events ...............................................................................................9 1.3.4. Egress.................................................................................................................12 1.4. LATENT ALPHAHERPESVIRUS INFECTION ......................................................................19 1.4.1. Introduction ........................................................................................................19 1.4.2. Establishment of latency......................................................................................21 1.4.2.1. Neuronal properties creating a favorable environment for establishment of latency ......................................................................................................................21 1.4.2.2. Role of the immunity during establishment of latency ..................................24 1.4.2.3. Alphaherpesvirus properties promoting latency ............................................29 1.4.2.4. Does virus replication preceed latency establishment?..................................33 1.4.3. Maintenance of latency .......................................................................................34 1.4.3.1. Continuous presence of components of the adaptive immune response in TG during maintenance of latency ..................................................................................34 1.4.3.2. Role of CD8+-T cells during maintenance of latency ....................................36 1.4.3.3. Gene silencing during maintenance of latency ..............................................39 1.4.4. Reactivation from latency....................................................................................39 1.4.4.1. Initiation of a lytic infection differs from reactivation from latency ..............40 1.4.4.2. Fate of neurons and newly produced virus upon successful reactivation .......44 1.4.5. Conclusion..........................................................................................................46 1.5. AIMS OF THE THESIS....................................................................................................47 CHAPTER 2: A HOMOLOGOUS IN VITRO MODEL TO STUDY INTERACTIONS BETWEEN ALPHAHERPESVIRUSES AND TRIGEMINAL GANGLION NEURONS....49 CHAPTER 3: ALPHAHERPESVIRUS GLYCOPROTEIN D INTERACTION WITH SENSORY NEURONS TRIGGERS FORMATION OF VARICOSITIES THAT SERVE AS VIRUS EXIT SITES ............................................................................................................59

CHAPTER 4: TRANSFECTION OF PSEUDORABIES VIRUS GLYCOPROTEIN D IN NECTIN-1 EXPRESSING CHO CELLS RESULTS IN RHO GTPASE-DEPENDENT FORMATION OF FILOPODIA-LIKE STRUCTURES .......................................................79 CHAPTER 5: INTERFERON ALPHA INDUCES ESTABLISHMENT OF A LATENCYLIKE STATE OF ALPHAHERPESVIRUS INFECTION IN TRIGEMINAL GANGLION NEURONS IN VITRO..........................................................................................................97 CHAPTER 6: GENERAL DISCUSSION .........................................................................115 CHAPTER 7: SUMMARY – SAMENVATTING ............................................................129 CHAPTER 8: REFERENCES...........................................................................................143 Curriculum vitae ....................................................................................................................183 Dankwoord ............................................................................................................................187

List of abbreviations APP

amyloid precursor protein

BoHV

bovine herpesvirus

CBP

CREB binding protein

CGRP

calcitonin gene-related peptide

CHO

Chinese hamster ovary

CNS

central nervous system

CREB

cAMP response element-binding

CTL

cytotoxic T lymphocyte

DNA

deoxyribonucleic acid

dpi

days post infection

DRG

dorsal root ganglion

E

early

EHV

equine herpesvirus

ER

endoplasmatic reticulum

FITC

fluorescein isothiocyanate

gB,gC,...

glycoproteinB, glycoproteinC

HCF

host cell factor

hpi

hours post infection

HSV

herpes simplex virus

HVEM

herpes virus entry mediator

ICER

inducible cAMP early repressor

ICP

infected cell protein

IE

immediate early

IFN

interferon

IL

interleukin

ILTV

infectious laryngotracheitis virus

IRF

interferon response factor

ISH

in situ hybridisation

L

late

LAT

latency associated transcript

MAPK

mitogen activated protein kinase

MHC

major histocompatibility complex

MTOC

microtubule organizing center

ND10

nuclear domain 10

NGF

nerve growth factor

NO

nitric oxide

NOS

nitric oxide synthetase

OAS

2’,5’-oligoadenylate synthetase

PCR

polymerase chain reaction

PKR

double stranded RNA-activated protein kinase

PML-NB

promyelocytic leukaemia nuclear body

PNS

peripheral nervous system

PRV

pseudorabies virus

RNA

ribonucleic acid

RT

reverse transcriptase

SCG

superior cervical ganglion

TCR

T cell receptor

TG

trigeminal ganglion

TK

thymidine kinase

TNF

tumor necrosis factor

TR

Texas Red

UL

unique long

US

unique short

VZV

varicella zoster virus

wt

wild type

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Chapter 1: Introduction 1.1. Pseudorabies virus, an alphaherpesvirus 1.2. Trigeminal ganglion neurons 1.3. Productive alphaherpesvirus infection in neurons and subsequent viral spread 1.4. Latent alphaherpesvirus infection 1.5. Aims of the thesis

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1.1. Pseudorabies virus, an alphaherpesvirus 1.1.1. Classification and introduction Pseudorabies virus (PRV), also known as Aujeszky’s disease virus or suid herpesvirus 1, belongs to the family of the Herpesviridae. All herpesviruses share some biological characteristics. They all encode their own enzymatic machinery for the nucleic acid metabolism and DNA synthesis, the capsid formation and encapsidation takes place in the nucleus and they all undergo a latent phase in their life cycle (Roizman & Pellet, 2001). Based on their biological properties and genome content and organisation, most herpesviruses can be subdivided into three major subfamilies: Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae. These subfamilies differ in the cell type where latency is established and the length of their productive replication cycle. Alphaherpesviruses, to which PRV belongs, have the broadest host range, replicate very rapidly in cell cultures and establish latency mainly in neurons of sensory ganglia. Betaherpesviruses have the most restricted host range, the slowest replication rate that is often accompanied by cell enlargements and establish latency in a number of tissues and cells, including secretory glands, kidneys and lymphoreticular cells. Gammaherpesviruses have a restricted host spectrum and establish latency in lymphoid tissue. Three human alphaherpesviruses are known: herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella zoster virus (VZV). Several animal pathogens important to agriculture also belong to the Alphaherpesvirinae including PRV, bovine herpesvirus 1 and 5 (BoHV-1 and -5), equine herpesvirus 1 and 4 (EHV-1 and -4), ovine herpesvirus and avian herpesviruses such as Marek’s disease virus and infectious laryngotracheitis virus (ILTV). Based on molecular criteria and sequence analysis, the subfamily of the Alphaherpesvirinae is subdivided into the genera Simplexvirus, Varicellovirus, Mardivirus and Iltovirus (International Commitee of Taxonomy of Viruses, 2004). PRV has been classified to the Varicellovirus genus together with VZV, BoHV-1, EHV-1 and EHV-4. HSV-1 and -2 are members of the Simplexvirus genus. PRV is the causative agent of Aujeszky’s disease in pigs (Aujeszky, 1902). Symptoms observed after infection of pigs with PRV depend on the age of the animals. Infection of young piglets results in mortality by central nervous system disorders, whereas older animals develop respiratory problems and reproductive failure like abortion and mummification (Wittmann et al, 1980; Pensaert et al, 1989). Pigs are considered to be the natural reservoir of

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PRV because they can survive a PRV infection. PRV is however also able to infect most other mammals, except higher primates, which is almost invariably associated with central nervous system disorders and death of the animal. Because of the succesful development and use of marker vaccines, PRV has been eradicated in significant parts of the United States and Europe (Mettenleiter, 2000). This, however, has not let to a lack of interest in research on PRV. Because of the significant homology between different alphaherpesviruses, PRV is frequently used as a model to study the biology of members of this subfamily (Pomeranz et al, 2005), especially of the human alphaherpesviruses which are difficult to study in their natural host. 1.1.2. Virion structure PRV virions, like all herpesvirus virions, are composed of four structural elements (Figure 1). The inner core of the virus particle consists of a double stranded lineair DNA molecule of approximately 143 kbp. The genome consists of 2 covalently bound segments, a unique long and a unique short segment, both flanked by an internal and a terminal repeat sequence. These two regions can invert relative to each other, thereby giving rise to two different DNA molecules that differ in the orientation of their DNA sequence (Ben-Porat et al, 1983; Whitley & Roizman, 2001; Klupp et al, 2004). The PRV genome contains 72 ORF’s and encodes 70 different proteins which all have orthologs in other alphaherpesviruses (Klupp et al, 2004; Pomeranz et al, 2005). The genome is encapsulated by an icosahedral capsid consisting out of 162 capsomers that is initially formed around a scaffold that is proteolytically cleaved and destroyed during the incorporation of the DNA. The capsid together with the DNA is called the nucleocapsid (Mettenleiter, 2000). This nucleocapsid is embedded in an amorphous, electron dense protein layer called the tegument that seems to consist out of an inner layer tightly associated with capsid proteins and a more assymetrically organized, heterogeneous outer layer beneath the viral envelope (Grunewald et al, 2003). The viral envelope forms the outermost layer of the virus particle and is a lipid bilayer membrane that originates from intracellular membranes of the trans-Golgi network (Mettenleiter, 2000). The envelope is decorated with 16 different viral (glyco)proteins, e.g. gB, gC, gD, gK, gE-gI, gH-gL, gM-gN and US9 (Granzow et al, 2001). These membrane proteins mainly function in virus entry, egress and cell-to-cell spread.

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Figure 1. Schematic representation of a PRV virion (A) and a linear map (B) and crystal structure of gD (C). (B and C were adapted from Campadelli-Fiume et al, 2007)

1.2. Trigeminal ganglion neurons 1.2.1. Anatomical structure Trigeminal ganglion neurons are sensory neurons belonging to the somatic part of the peripheral nervous system and are responsible for both touch/position and pain/temperature sensation in the face. The trigeminal ganglion (TG) contains the cell bodies of these sensory neurons. They are pseudo-unipolar neurons with one branch connecting the cell body to the central nervous system (CNS) and another one going to the periphery (Firbas et al, 1995). The sensory neurons are all part of the fifth cranial nerve (also known as the trigeminal nerve). Besides sensory nerve fibers, the fifth cranial nerve also contains some motor nerve fibers which also pass through but do not have their cell bodies in the TG. From the TG, a single large sensory root enters the brainstem at the level of the pons and connects the sensory neurons of the TG to the trigeminal nucleus which is part of the CNS and extends throughout the entire brainstem. Adjacent to this sensory root, a smaller motor root also enters the pons leading the motor nerve fibers to their cell bodies located in the motor nucleus of the fifth nerve which lies near the main trigeminal nucleus. Towards the facial aria, 3 branches of the trigeminal nerve originate from the TG. i) The opthalmic nerve that carries sensory information in part from the upper eyelid, the conjunctiva and the cornea of the eye, the nose and the nasal mucosa. ii) The maxillary nerve that carries sensory information in part from the lower eyelid, the nasal mucosa, the upper lip and the cheek. iii) The mandibular nerve that carries sensory information in part from the lower lip and the tongue. The motor branches of the trigeminal nerve are distributed in the mandibular nerve and are involved in motor

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functions as biting, chewing and swallowing (Jenkins, 1978; Paxinos & Mai, 2004; Siegel & Sapru, 2006). The TG consists of neurons, non-neuronal satellite cells, fibroblasts and endothelial cells. The ratio of neurons to non-neuronal cells in ganglia is approximately 1 to 100 (Laguardia et al, 2000). Human TG have been shown to contain on average 27400 neurons per ganglion, compared to 35300 neurons per ganglion for rats (Lagares & Avendano, 2000; Laguardia et al, 2000). The mean diameters for a human TG neuron and a non-neuronal cell were estimated to be 60 µm and 8 µm respectively (Melling et al, 2001). Primary sensory neurons from the TG are a diverse population of cells that can be classified according to their cellular morphology, physiological response properties and patterns of gene expression. TG neurons can be classified as large, medium or small (Alvarez et al, 1991; Lagares & Avendano, 2000). Others have grouped them in separate populations based on calcitonin gene-related peptide (CGRP) or substance P expression (Alvarez et al, 1991; LaVail et al, 1991; Margolis et al, 1992) and others grouped them based on the presence of cell-surface glycoconjugates, oligosaccharide antigens or lectins recognized by monoclonal antibodies SSEA3, LD2, BSIL4, LA4, A5 and KH10 (Alvarez et al, 1991; Margolis et al, 1992; Margolis et al, 2007). As mentioned, the sensory nerves of the fifth nerve carry both touch/position and pain/temperature information. Pain/temperature information is carried by unmyelinated nerve fibers while touch/position information is carried by myelinated nerve fibers. The myelin sheath is formed by Schwann cells that wrap around the axon shaft. It protects the axons from damage and serves as an insulator to speed conduction of electrical pulses. Along the axon, there are several breaks in the continuity of the myelin sheath which are called nodes of Ranvier. These are strongly enriched in voltage gated Na+ and K+ ion channels and form places were the axon is in contact with the extracellular fluid (Paxinos & Mai, 2004; Siegel & Sapru, 2006). After crossing the basement membrane, axons of TG neurons are no longer covered by a myelin sheath and these unmyelinated nerve endings running in between epithelial cells are prone for the formation of pre-synaptic boutons, so-called varicosities (Kondo et al, 1992; Ibuki et al, 1996). 1.2.2. Primary target cells for different alphaherpesviruses For several alphaherpesviruses like HSV-1, PRV and BoHV-1, neurons of the TG are of special interest because they are characterized as the predominant site for the establishment of latent infections (Baringer & Swoveland, 1972; Stevens et al, 1972; Cook et al, 1974;

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Gutekunst et al, 1980; Ackerman et al, 1982; Croen et al, 1987). The virus reaches the TG neurons via the axon endings that innervate the place of primary virus infection: the mucosa and epithelial cells of the upper respiratory tract (Baskerville, 1973; Miry & Pensaert, 1989). After reaching the neuronal nucleus, alphaherpesviruses can either start a productive infection with the formation of new infectious virus particles or enter a latent state of infection where no new virus particles are produced (Roizman & Knipe, 2001). The course of a productive infection in neurons will be reviewed in section 1.3 while the factors involved in establishment and maintenance of and reactivation from a latent infection will be reviewed in section 1.4.

1.3. Productive alphaherpesvirus infection in neurons and subsequent viral spread Although a part of the data that will be described in this section is specific for PRV, most information comes from studies using HSV-1, the best characterized virus within the Alphaherpesvirinae. 1.3.1. Entry Upon infection of a host, primary replication of PRV occurs in the mucosa of the upper respiratory tract. After this primary replication, free PRV virions enter into nerve endings of trigeminal ganglion neurons that are present in these mucosa (Figure 2).

Figure 2. Schematic representation of the entry of a PRV virion at the axon terminus and subsequent intra-axonal retrograde transport towards the nucleus. Entry of PRV into the nerve endings begins with receptor binding (1) followed by fusion between the viral envelope and the plasma membrane (2) and subsequent release of the nucleocapsid and associated tegument into the axoplasm (3). Retrograde transport of the

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nucleocapsids towards the nucleus is mediated by dynein motor proteins that transport nucleocapsids along microtubules (4). (Adapted from Tomishima et al, 2001).

The entry of PRV and HSV-1 in nerve endings is mediated by fusion between the viral envelope and the axonal plasma membrane. Five glycoproteins present in the viral envelope are involved in this entry process, i.e. gB, gC, gD, gH and gL (Pomeranz et al, 2005). First, a labile interaction occurs between gB and/or gC and heparan sulfate proteoglycans present on the cell surface. Although this binding significantly enhances the efficiency of infection, it is not absolutely essential (Mettenleiter et al, 1990; Karger et al, 1995; Immergluck et al, 1998; Shukla & Spear, 2001; Spear & Longnecker, 2003). A subsequent stable binding is established by binding of gD to its cellular receptor (Karger & Mettenleiter, 1993). Several receptors for gD have been characterised which belong to 3 different classes. A first receptor is the herpesvirus entry mediator (HVEM) which belongs to the tumor necrosis factor receptor family. Nectin-1 and -2 are two receptors belonging to the immunoglobulin superfamily and another type of receptor consists of modified heparan sulfate originating from specific sulfation by certain 3-O-sulfotransferases (Shukla & Spear, 2001; Spear & Longnecker, 2003). Nectin-1 has been suggested to serve as the predominant gD receptor on sensory neurons for both HSV and PRV (Haarr et al, 2001; Mata et al, 2001; Milne et al, 2001; Richart et al, 2003; Simpson et al, 2005; Ono et al, 2006). The modified heparan sulfate however can not be excluded seen the recent report that trigeminal ganglion neurons express important isoforms of 3-O-sulfotransferases that produce 3-O-sulfated motifs that can be used by gD as receptor (Lawrence et al, 2007). The stable binding of gD to its receptor initiates the fusion process leading to fusion of the viral envelope with the plasma membrane resulting in the release of viral capsids in the cytoplasm. The exact mechanism of fusion has not yet been clarified but it has been proposed that gD binding to its receptor results in a conformational modification of gD by which it is able to interact with and/or recruit other viral fusion proteins, e.g. gB and the gH-gL complex (Carfi et al, 2001; Cocchi et al, 2004, Zago et al, 2004, Connolly et al, 2005). Glycoproteins gB and gH-gL are necessary for fusion because absence of one of them abolishes viral entry (Rauh & Mettenleiter, 1991; Rauh et al, 1991; Peeters et al, 1992a,b; Klupp et al, 1997; Turner et al, 1998; Mata et al, 2001; Nicola & Straus, 2004; Gianni et al, 2006; Subramanian & Geraghty, 2007; Reske et al, 2007).

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1.3.2. Transport to the nucleus During fusion with the plasma membrane, virions loose their envelope and part of the tegument dissociates from the capsid by cytoplasmic phosphorylation events mediated by both virion associated and cellular kinases (Morrison et al, 1998). Recent studies have shown that tegument proteins VP11/12, VP13/14, VP16, VP22 and UL11 quickly dissociate from the capsid, while UL36 (VP1/2), UL37 and US3 remain associated (Granzow et al, 2005; Luxton et al, 2005). Because genome replication occurs in the neuronal nucleus, capsids containing the DNA have to be transported over long distances (>10 cm) from the axon termini to the cell body where the nucleus is located (Batterson et al, 1983; Lycke et al, 1984 and 1988; Sodeik et al, 1997). To travel these long distances, virions use the cellular microtubule-mediated fast-axonal transport machinery (Figure 2). To this end, they have been proposed to bind to dynein, a motor protein that is able to transport cargo on microtubules from the plus-end (located at the axon terminus) towards the minus-end (located at the microtubule organizing center (MTOC) near the nucleus). By binding to dynein, the virions are retrogradely transported towards the nucleus with an average rate of 1.3 µm/s (Kristensson et al, 1986; Topp et al, 1994; Sodeik et al, 1997; Bearer et al, 2000; Smith et al, 2001 and 2004; Döhner et al, 2002). Both the capsid protein VP26 and UL34, a protein that is thought to be primarily a membrane protein but that is also at least partly present in the tegument, of HSV-1 have been shown to interact with dynein and were therefore proposed to be candidates to engage dynein to capsids (Purves et al, 1992; Ye et al, 2000; Douglas et al, 2004). The role of both proteins in retrograde transport has however been challenged. UL34 is absent from mature PRV virions (Fuchs et al, 2002b) and is not essential during HSV-1 and PRV infection of cultured cells (Klupp et al, 2000; Roller et al, 2000) while virus mutants lacking VP26 were still transported to the nucleus and this transport relied on dynein (Antinone & Smith, 2006; Dohner et al, 2006). This suggests that other receptors for dynein are encoded by HSV-1. The observation that fluorescent gfplabeled capsids also moved in the anterograde direction after entry show that PRV probably also associates with a plus-end directed motor protein at this stage of infection (Smith et al, 2004). After the retrograde transport of capsids to the nucleus, they bind to the nuclear membrane and associate with nuclear pore complexes, mediated by cellular importin β and the VP1/2 tegument protein (Ojala et al, 2000). After the formation of the docking complex, viral DNA

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is rapidly released into the nucleus (Batterson et al, 1983; Sodeik et al, 1997; Ojala et al, 2000). 1.3.3. Intranuclear events As described in 1.2.2, two routes of infection can be followed after viral DNA enters the neuronal nucleus. Either a productive infection cycle leading to formation of new virus particles is initiated or a latent infection is established. This paragraph will describe the events occuring during a productive infection while indications of mechanisms leading to a latent state of infection will be described in section 1.4.

Figure 3. Schematic representation of the PRV replication cycle in the neuronal cell body. When nucleocapsids arrive in the cell body, viral DNA is released into the nucleus through the nuclear pore complex (1). Upon entry of viral DNA in the nucleus, viral DNA replication (2) and transcription (3) are initiated followed by translation and new viral protein synthesis in the cytoplasm (4). Some newly produced proteins are transported back to the nucleus where procapsid formation (5) and DNA packaging into these capsids (6) occurs. The egress of mature nucleocapsids occurs through a process of primary envelopment and de-envelopment at the nuclear membrane, resulting in release of naked nucleocapsids in the cytoplasm (7). During egress of mature virions via the traditional model, naked nucleocapsids acquire an inner tegument in the cytoplasm (8) which is followed by budding in the trans-Golgi network, thereby obtaining their outer tegument and final envelope (9). Enveloped virus particles are released from the neuronal cell body via vesicle-mediated (10) exocytosis (11). (Adapted from Tomishima et al, 2001 and Pomeranz et al, 2005).

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Upon release of the viral genome in the nucleus (Figure 3), the DNA is treated as a foreign DNA molecule. It has been shown that at least a portion of HSV-1 genomes becomes rapidly associated with histones after nuclear entry, bringing the DNA in a chromatin configuration (Kent et al, 2004). Histones can undergo post-translational modifications (e.g. acetylation, methylation and phosphorylation) of their terminal tail which can lead to an open chromatin configuration (transcriptional activity) or to a condensed chromatin state (gene silencing) (Grewal & Moazed, 2003; Iizuka & Smith, 2003). It has been reported that the tegument protein VP16 recruits histone acetyltransferases and chromatin remodelling enzymes to virus IE promoters and that it may function to exclude histone deposition at actively transcribed promoters (Herrera & Triezenberg, 2004). Similarly, another tegument protein, VP22, has been shown to interact with cellular proteins to prevent histone deposition onto DNA (van Leeuwen et al, 2003). These data support the view that viral proteins try to overcome silencing of the viral genome after entry in the nucleus. Whether VP16 and VP22 are able to perform this function during alphaherpesvirus infection of neurons is uncertain, especially when considering the long distance these proteins have to travel to reach the nucleus after they have become dissociated from the capsid immediatly upon entry in the cell. Repression of gene transcription by assembly of a repressed chromatin structure and histone deacetylation can also be linked to the observed association of incoming viral DNA to promyelocytic leukaemia nuclear bodies (PML-NB, also known as ND10), which appears to be an antiviral mechanism of the cell in response to several DNA viruses (reviewed by Everett & ChelbiAlix, 2007). These ND10 are small nuclear substructures consisting of several proteins like PML, Sp100 and SUMO and some components that are involved in chromatin metabolism and remodelling like ATRX, hDaxx and others (Luciani et al, 2006). ND10 structures are also commonly associated with sites of DNA damage (Dellaire et al, 2006; Boe et al, 2006). It has long been thought that incoming viral DNA was sequestered to existing ND10 structures (Maul et al, 1996; Maul, 1998) but recent indications indicate that de novo ND10 structures are formed in association with viral genome complexes during the initial stages of HSV-1 infection (Everett & Murray, 2005; Everett et al, 2004). After the association of the viral genome with ND10, the first viral proteins are transcribed. As for all herpesviruses, HSV-1 and PRV gene expression is characterised by a tightly regulated temporal pattern of expression of three gene classes: immediate early (IE) genes, early (E) genes and late (L) genes (Roizman and Knipe, 2001). PRV expresses only one protein with IE kinetics, i.e. IE180, the orthologue of ICP4 of HSV-1 (Cheung et al, 1989; Martin et al, 1990; Wu & Wilcox, 1991). HSV-1 expresses another four proteins with IE

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kinetics, ICP0, ICP22, ICP27 and ICP47. The first three of them have an orthologue in the PRV genome in which they are expressed with early kinetics (Pomeranz et al, 2005). For HSV-1, transcription of IE genes is activated by the tegument protein VP16 forming a complex with cellular HCF and Oct-1. This complex binds to a specific TAATGARAT sequence present in all HSV-1 IE promoters, thereby initiating transcription (Roizman & Knipe, 2001). Similar to IE180 of PRV, ICP4 is the first HSV-1 protein to be produced upon infection and ICP4 foci can be observed juxtaposed to ND10 at early times post infection. For at least some of these ICP4 foci, it was shown that they contained the viral genome (Everett et al, 2004). During lytic infection of HSV-1, ND10 are destroyed by ICP0, another IE protein that contains a RING finger domain that possesses ubiquitin E3 ligase activity and that induces the degradation of PML and Sp100 in a proteasome dependent manner (Boutell et al, 2002). EP0, the PRV orthologue of ICP0, has also been shown to contain the RING finger domain and was found to be able to destroy ND10 in transfection experiments (Parkinson & Everett, 2000 and 2001). Once IE genes are transcribed in adequate amounts, they transactivate the expression of E genes which primarily encode proteins involved in DNA replication. These proteins together with the viral DNA localize in so-called replication compartments in the nucleus. These replication compartments originate from some of the ICP4 foci and represent sites where DNA replication takes place (Quinlan et al, 1984; de Bruyn Kops et al, 1994 and 1998; Lukonis et al, 1997; Lukonis and Weller, 1997; Liptak et al, 1996; Everett et al, 2004; Taylor et al, 2003; Knipe et al, 1987). Importantly, all observations about the role and fate of ND10 early after entry of DNA in the nucleus were done in non-neuronal cells. Their role in infection of neurons has to be elucidated, especially since ND10 do not seem to be generally present in neurons and neuronal cell lines (Lam et al, 1995; Negorev and Maul, 2001; Villagra et al, 2004). An initial step in the replication of herpesvirus DNA is the formation of circular molecules from the infected linear genome. Genome circularization of PRV most likely occurs by blunt end ligation of the free ends and does not require any viral protein synthesis. The circular genomes serve as the template for DNA synthesis and allow the genome to be replicated by a rolling circle mechanism. The latter process produces replicated DNA in the form of long linear concatemeric DNA molecules that are cleaved into unique length genomes upon encapsidation (Ben-Porat & Kaplan, 1984). Recently however, Jackson & DeLuca (2003) provided strong evidence that circularisation of the viral genome did not occur during productive HSV-1 infection. They showed that circularization was inhibited by expression of ICP0 and therefore they hypothesized that circularisation occurs during latent infection, rather

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than during productive infection. Since the ICP0 orthologue of PRV, EP0, is expressed as an early rather than an immediate early protein, it is possible that the PRV genome is circular during productive infection since EP0 may be expressed after circularisation already has occured. DNA replication triggers the transcription of L genes. These late genes mainly encode structural proteins. One group of L proteins are important for capsid formation. The final HSV-1 capsid has an icosahedral structure consisting of 150 hexons and 12 pentons and is formed around a procapsid structure built with VP5, pre-VP22a, VP19c and VP23 (Homa & Brown, 1997; Spencer et al, 1998; Newcomb et al, 2001). After removal of pre-VP22a from the procapsid by VP24 (Walters et al, 2003), free space in the capsid is generated that is filled with viral DNA during encapsidation. Encapsidation requires two linked events, cleavage of the concatemeric DNA in monomeric units and packaging of these monomeric genomes in the capsids. Both processes rely on ICP18.5 (Mettenleiter et al, 1993; Ladin et al, 1980) and result in the formation of mature nucleocapsids. 1.3.4. Egress The egress of mature nucleocapsids from the nucleus starts by a budding process at the inner nuclear membrane, resulting in capsids surrounded by a primary tegument and envelope in the perinuclear space. A key role in this primary envelopment step of both HSV-1 and PRV is attributed to viral proteins UL31 and UL34 which can promote vesicle formation in the perinuclear space, even without the presence of any other viral protein (Klupp et al, 2000: Reynolds et al, 2001 and 2004; Mettenleiter, 2002b; Klupp et al, 2007). The envelope of the perinuclear primary enveloped virions then fuses with the outer leaflet of the nuclear membrane, resulting in loss of the primary envelope and release of capsids into the cytoplasm. The exact mechanism of this de-envelopment step is not yet known, but the US3 serine/threonine protein kinase seems to play a major role since mutations in the US3 orthologue of PRV, HSV-1 and Marek’s disease virus result in accumulation of perinuclear enveloped virions (Klupp et al, 2001; Mettenleiter, 2002b and 2006; Reynolds et al, 2002, Granzow et al, 2004; Schumacher et al, 2005). For PRV, US3 is the only tegument protein that is found in both primary and mature virions (Granzow, 2004), while for HSV-1, both US3 and VP16 are found in both forms of enveloped virions (Reynolds et al, 2002; NaldinhoSouto et al, 2006). The naked cytoplasmic nucleocapsids obtain their final tegument in the cytoplasm in what is believed a 2-step tegumentation process. The inner tegument is

Introduction 13 ____________________________________________________________________________________________________

presumably formed by UL36 that contacts the capsid and which has been shown to interact with UL37. US3 has also been shown to be part of the capsid-associated tegument (Zhou et al, 1999, Klupp et al, 2002; Granzow et al, 2004; Fuchs et al, 2004). Other tegument proteins like UL11 and VP22 have been shown to accumulate at membranes of the trans-Golgi, the secondary envelopment site. For both HSV-1 and PRV it has been shown that these proteins interact with intracytoplasmatic domains of glycoproteins that are embedded in trans-Golgi membranes (Brack et al, 1999 and 2000; Fuchs et al, 2002a; Chi et al, 2005; Farnsworth et al, 2003 and 2007). The complete mechanism of secondary envelopment is not yet understood, but probably a network of protein-protein interactions between the mentioned and other tegument proteins and nucleocapsid proteins and cytoplasmatic domains of glycoproteins cause the tegumented capsid to bud in the trans-Golgi, thereby obtaining its final envelope (Mettenleiter, 2006). Subsequent release of infectious virus particles from the neuronal cell body occurs via exocytosis of virion-containing vesicles, similar to viral egress from non-neuronal cells (Granzow et al, 2001; Miranda-Saksena et al, 2000 and 2002). In neurons however, HSV-1 and PRV do not only spread from the cell body, but they are also transported via axons to spread to the periphery where they cause recurrent disease or they are able to perform transneuronal spread to synaptically connected neurons of the peripheral and central nervous system (Roizman & Knipe, 2001; Enquist, 2002; Feierbach et al, 2007). Many questions still remain about the anterograde transport and subsequent spread of newly formed alphaherpesvirus virions. Just like retrograde transport, anterograde transport has been shown to rely on microtubule-based fast axonal transport. The anterograde transport was shown to be bi-directional with a net movement towards the axon ending with an average velocity of 2.0 µm/s, indicating that the virus recruits both minus- and plus- end directed motor proteins (Smith et al, 2001; Lee et al, 2006). In contrast to the consensus that alphaherpesviruses use microtubule-based fast axonal transport to move in the anterograde direction, contradictory data exist about the form in which virions are transported to the axon endings. Two models have been proposed, the traditional model and the subvirion model (Figure 4), each being supported by a set of well-documented data.

14 Chapter 1 ____________________________________________________________________________________________________

Figure 4. Schematic representation of PRV egress at the axon terminus. In the traditional model of egress, enveloped PRV particles are transported in the axon inside a transport vesicle. Fusion of the transport vesicle with the plasma membrane at the axon terminus results in release of PRV (A). In the subvirion model, capsids and tegument are transported separately from glycoprotein-containing vesicles. Release of infectious virus from the axon terminus occurs after glycoprotein-containing vesicles fuse i) with the axolemma followed by budding of capsids through the envelope protein containing membrane (B1) or ii) with endosomes present in the axon terminus followed by budding of the capsid in the envelope protein containing endosome and subsequent release via exocytosis (B2). (Adapted from Tomishima et al, 2001).

In the traditional model of anterograde spread, alphaherpesviruses become fully assembled in the neuronal cell body by budding of capsids into the trans-Golgi whereby enveloped virions are acquired and subsequently, these enveloped virions are carried in transport vesicles from the cell body to axon termini (Kristensson et al, 1974; Lycke et al, 1988; Card et al, 1993; Tomishima et al, 2001). This model closely resembles egress from non-neuronal cells. The transport of vesicles derived from the trans-Golgi network by fast-axonal microtubule-based

Introduction 15 ____________________________________________________________________________________________________

transport in neurons is well established and is very important for the physiological integrity of this cell type, e.g. the transport of neurotransmitter containing synaptic vesicles along the axon to places where neurons synapse with other cells (Goldstein & Yang, 2000). Since PRV and HSV-1 have not been found to encode motor proteins for transport on microtubules, they have to rely on the cellular plus-end motor protein kinesin for transport to the axon terminus. How the virus containing vesicles bind kinesin is not entirely clear. One possibility is that the amyloid precursor protein (APP), a known cellular motor protein receptor which is present in the membranes of the Golgi, causes the virus containing vesicles to bind to kinesin, followed by anterograde transport (Satpute-Krishnan et al, 2003). Another possibility is that one or more viral tegument proteins or the cytoplasmic domain of envelope proteins that are present on the outside of the virus containing vesicles bind directly or indirectly to cellular motor proteins. A first line of support for the traditional model is that enveloped PRV and HSV-1 virions in vesicles have been detected by electron microscopy in axons of different kinds of neurons, e.g. in sympathetic neurons of the rat superior cervical ganglion (SCG) and in sensory neurons of human and rat dorsal root ganglia (DRG) (Marchand et al, 1986; Lycke et al, 1988; Card et al, 1993; Ch’ng & Enquist, 2005; del Rio et al, 2005). Another argument in favour of this model is that infectious PRV could be isolated from mid-axons of trigeminal ganglion neurons of pigs infected by intranasal inoculation (Kritas et al, 1994a,b and 1995). Because it has been clearly established that alphaherpesviruses loose their envelope during entry of neurons what corresponds to a non-infectious state, the former arguments can not be due to visualisation or isolation of retrogradely transported virus (Lycke et al, 1984 and 1988, Card et al, 1993; Tomishima & Enquist, 2002). Recent studies have been based on dualfluorescent PRV viruses, in which the genes encoding a capsid protein and a tegument protein were fused to genes encoding different fluorescent proteins. These experiments were done in living neurons and gathered observations supporting the traditional model of anterograde viral spread. Time lapse imaging of these dual-fluorescent viruses in living axons of cultured chick DRG neurons showed that the capsid signal always moved together with the signal of the tegument proteins in the anterograde direction. Together, these data are consistent with a model in which the capsid acquires a full tegument before initiation of axonal transport (del Rio et al, 2005; Luxton et al, 2005). The remaining possibility that the capsid transports together with the tegument, but separate from the envelope, was ruled out by another study using dual-fluorescent PRV viruses, in which a capsid protein and the envelope protein gD were tagged with a different fluorescent protein. Again, the capsid almost always moved together with the envelope in the anterograde direction whereas progeny viral particles that

16 Chapter 1 ____________________________________________________________________________________________________

lacked a membrane were immediatly directed back to the cell body (Antinone & Smith, 2006). In further proof of the traditional model, the same study also showed that capsids were cotransported with a fluorescent signal comming from DiOC16, a lipophilic dye, thereby suggesting that egressing capsids were membrane bound. Whereas capsid transport without tegument or envelope was almost never detected, transport of tegument and envelope proteins alone without capsids was commonly observed (Luxton et al, 2005; Antinone & Smith, 2006). A second model for the anterograde spread of alphaherpesviruses was proposed for the first time in 1994 by coworkers of the Cunningham lab and is referred to as the subvirion model. In this model, envelope proteins are thought to be transported to the axon terminus in glycoprotein-containing vesicles (originating from the Golgi in the neuronal cell body) but separately from capsids and tegument which do not use vesicles for their anterograde transport. The initial evidence for this model came from an electron microscopy study in which HSV-1 particles moving in the anterograde direction could only be detected as unenveloped capsids in axons of human fetal DRG neurons (Penfold et al, 1994). Further immunoelectron microscopy showed unenveloped capsids immunolabeled for VP5 and surrounded by tegument proteins (VP16) adjacent to microtubules, whereas glycoproteins and other tegument proteins were observed within axonal vesicles without capsids in human and rat DRG neurons (Holland et al, 1999; Miranda-Saksena et al, 2000). Another immunoelectron microscopy study on cross sections of axons in the optic tracts of infected mice at 5 days post injection into the posterior chamber of the eye showed the presence of unenveloped capsid-like structures positive for viral DNA surrounded by a halo of diffuse, electron-dense material, presumably being the tegument (La Vail et al, 2005). More evidence supporting this model was gathered by immunofluorescence microscopy studies in different kinds of neurons in which PRV and HSV-1 nucleocapsids in axons were often found separately from vesicles containing viral glycoproteins (Miranda-Saksena et al, 2000; Smith et al, 2001; Enquist et al, 2002; Potel et al, 2003; Snyder et al, 2006 and 2007). The observation that the US9 protein of PRV was necessary for the localisation of glycoproteins into the axon, but not for capsid and tegument proteins seemed also in favor of the subvirion model (Tomishima & Enquist, 2001). However, the same group recently reported that axons of PRV-US9null infected neurons contained not only no glycoproteins but also no capsids, what is again in favor of the traditional model of anterograde spread (Lyman et al, 2007). A somewhat similar observation was recently done for HSV-1 (LaVail et al, 2007). However, in this in vivo study, US9 was found to be necessary for anterograde axonal transport of nucleocapsids but not for transport of the virus envelope. This remarkable difference was

Introduction 17 ____________________________________________________________________________________________________

suggested to be attributable to differences between both viruses and the neurons used. Another argument supporting the subvirion model is that the administration of brefeldin A, a product that disrupts the Golgi and thereby ablates vesicle formation, could not inhibit the anterograde transport of capsids in human DRG neurons (Miranda-Saksena et al, 2000). Other studies found however that brefeldin A inhibited both capsid and glycoprotein transport and that brefeldin A can not be used to distinguish between models of HSV axonal transport (del Rio et al, 2005; Snyder et al, 2006). One more argument that is used by some to support the subvirion model is that glycoproteins enter the axon earlier than the capsids after infection or that glycoprotein transport in the axon is not impaired when neurons are infected with HSV mutants that produce immature capsids that are retained in the nucleus (Miranda-Saksena et al, 2000; LaVail et al, 2003; Snyder et al, 2006). It was mentioned however before that the transport of glycoproteins and some tegument proteins without being associated with capsids is commonly observed (Luxton et al, 2005; Antinone & Smith, 2006) and therefore is not a convincing argument for the subvirion model. An interesting question related to the subvirion model is how capsids, possibly surrounded by a part of the tegument, bind to kinesin, the plus-end directed cellular motor protein, in the absence of a surrounding vesicle as in the traditional model. The observation that HSV-1 capsids that were only surrounded by the inner tegument, obtained from extracellular virions treated with triton-X and different KCl concentrations, can be transported on microtubules show that one of these tegument proteins must be able to engage to motor proteins and may as such support the subvirion model (Wolfstein et al, 2006). In this context, the HSV-1 tegument protein US11 was found to directly interact with the heavy chain of kinesin and was therefore suggested to be the protein that links capsids to kinesin (Diefenbach et al, 2002). The biological significance of this observation has to be established since many alphaherpesviruses do not encode a US11 homologue (Tomishima et al, 2001). Since capsids and glycoproteins are supposed to be transported separately towards the axon termini in the subvirion model, fully infective virions have to be assembled there, otherwise, surrounding cells could not become infected. Two alternative models for alphaherpesvirus assembly and egress from axon termini have been suggested (Figure 4B1 and 4B2). In a first model, transport vesicles containing envelope proteins fuse with the axolemma, subsequently followed by the budding of the capsids through this envelope protein containing plasma membrane, resulting in the release of an enveloped virus in the extracellular environment. In the second model, envelope protein containing vesicles fuse with endosomes present at the axon terminus, followed by the budding of the capsid in the envelope protein containing endosome, resulting in fully

18 Chapter 1 ____________________________________________________________________________________________________

assembled virions present in the endosome. Release of the virus then occurs by exocytosis (Tomishima et al, 2001). Till now, there is no answer to the question how all these arguments in favour of one of both models can be reconciled. Differences in viral strains and between alphaherpesviruses can play a role but it is unlikely that they could account for the use of such a completely different process of anterograde transport. It is more likely that there are quantitative differences in the relative importance of both models, in part due to the alphaherpesvirus and the neurons used (Cunningham et al, 2006). In many reports, both in favour of the traditional or the subvirion model, it is described that alphaherpesviruses are transported along microtubules to the axon termini where they leave the axon and infect surrounding cells. It has however been observed that glial cells surrounding the axon shaft also become infected (Ohara et al, 2001; Shimeld et al, 2001; Tomishima & Enquist, 2002). Retrograde transport of unenveloped virions (non-infectious) can not account for this observation, so newly formed virus has to be responsible for the infection of those cells (Lycke et al, 1984). Some looked at this observation as an extra argument for the traditional model of anterograde virus spread, suggesting that infectious enveloped virions are present in the axon and that those can leave the axon along the axon shaft to infect the glial cells (Kritas et al, 1994a,b and 1995; Shimeld et al, 2001). At first sight, the observation of infected glial cells appears to be inconsistent with the subvirion model since this model does not support assembly of infectious virions along the axon shaft. Ohara et al (2001) suggested that the glial cells became infected by virus that was released from the neuronal cell body and that diffused through the endoneurium (space outside the axon but within the nerve) by cell-to-cell spread. This hypothesis was proven very unlikely by a study using a gD negative mutant of PRV grown on a gD complementing cell line. This mutant produces no infectious extracellular virus but is capable to spread from cell-to-cell. It was shown that wild type PRV as well as gDnull mutants could spread from axons to closely apposed non-neuronal cells within the rat optic nerve but that no further spread occured from these cells. The restrictive capacity of Schwann cells to produce infectious virus was described before (Townsend & Collins, 1986; Wilkinson et al, 1999). Furthermore, cell-tocell spread, regarded as being slow by the limited cell size and the duration of a replication cycle, could theoretically not account for the observed infected cells at such a large distance away from the cell body (Tomishima & Enquist, 2002). The authors therefore suggested that viral envelope proteins may be transported separately from capsids and tegument, but that in this case, there must exist assembly and egress sites at scattered sites along the axon shaft as

Introduction 19 ____________________________________________________________________________________________________

well as at nerve terminals. This was in line with the results from a time lapse microscopy study of Smith et al (2001) showing that capsids not only accumulated at the nerve terminus but also, to a lesser extent, at sites along the axon shaft that were suggested to represent varicosities. The spread of PRV at scattered sites along the axon was further confirmed in vitro (Ch’ng & Enquist, 2005) and it was suggested that growth cones and varicosities, just as axon termini, could serve as assembly places for HSV-1 envelopment, followed by axonal egress from these structures by exocytosis (Saksena et al, 2006). From these data, it can be concluded that alphaherpesviruses not only leave axons at the axon terminus but can also exit at scattered sites along the axon shaft, suggested to be varicosities. This axonal egress can however be consistent with both models for anterograde viral transport.

1.4. Latent alphaherpesvirus infection 1.4.1. Introduction Despite

many

similarities,

some

properties

concerning

latency

differ

between

alphaherpesviruses. Therefore, this section will only discuss data obtained by studies with HSV-1, PRV and occasionally, BoHV-1. Latency is an important aspect of the lifecycle of a limited number of viruses, i.e. herpesviruses and retroviruses. It is a strategy of these viruses to stay present in their host during its entire lifetime without being eliminated by the host’s immune system. During a latent stage of infection, no new virus particles are produced but several specific stimuli can lead to periodic reactivation from this latent state and result in production of new virus particles that can give rise to recurrent disease symptoms. Neurons are the most important cell type for latent alphaherpesvirus infections. For HSV-1, PRV and BoHV-1, it has been shown that trigeminal ganglion neurons are the most predominant target cells for a latent infection while HSV-2 latent infections are mostly observed in dorsal root or sacral ganglion neurons (Baringer & Swoveland, 1972; Stevens et al, 1973; Baringer, 1974; Cook et al, 1974; Gutekunst et al, 1980; Ackerman et al, 1982; Croen et al, 1987). Latent VZV genomes are found in trigeminal, dorsal root and autonomic nervous system ganglia (Gilden et al, 1983 and 2001; Mahalingam et al, 1990). The latency/reactivation cycle can be subdivided in three phases. The first phase is the establishment of a latent infection during which the viral genome is directed to a dormant state after its entrance in the nucleus. Afterwards, the genome is kept in this dormant state

20 Chapter 1 ____________________________________________________________________________________________________

during the maintenance phase of latency. The viral genome can remain in this dormant state for a long time, but upon specific stimuli, it may proceed to the third phase, reactivation, which leads to new virus particle production. Since 1970, many studies have been undertaken to unravel the mechanisms controlling the latency/reactivation cycle. Some of the fundamental aspects of the latency/reactivation cycle begin to be understood but many of the molecular mechanisms controlling each phase remain to be clarified. Early studies showed that HSV-1 DNA extracted from the brainstem and TG of latently infected mice had no free ends, indicating that the genome is endless or circular during latency (Rock & Fraser, 1983 and 1985; Efstathiou et al, 1986). Furthermore, cesium chloride centrifugation of DNA from mice latently infected with HSV-1 showed that the viral DNA is extrachromosomal and exists as an episome (Mellerick & Fraser, 1987). Besides the status of the viral DNA during latency, it is accepted that during a latent HSV-1 and PRV infection, no viral proteins are produced (Roizman & Knipe, 2001). Many other data remain controversial and contradictory support has been found for many aspects of the latency/reactivation cycle. This will be reviewed in the following sections. Because the study of latency/reactivation of herpesviruses in humans is not possible in vivo, different model systems are used. In vivo studies are performed in small animal models like mice and rabbits and BoHV-1 is studied in cattle. Wild type as well as knock-out and transgene animals are used. Besides in vivo studies, an ex vivo explant model in which ganglia from latently infected mice are removed and cultured in vitro is also often used to study the maintenance and reactivation phase of latency. In both these models, latency is induced under physiologically relevant conditions in the animal. Besides these in vivo and ex vivo models, different in vitro models have been developed, consisting of primary neurons or continuous neuronal or non-neuronal cell lines quiescently infected with alphaherpesviruses. In these in vitro models, the quiescent state is generally induced by using chemical virus replication inhibitors, applying non-permissive conditions or by using replication deficient virus strains. All of these in vitro models are associated with several disadvantages and their relevance to in vivo human latency is often hard to predict (Preston, 2000). It is becoming increasingly clear that alphaherpesvirus latency is dependent on the interplay between neurons, virus and the immune system (Divito et al, 2006). In the following sections, the knowledge about the contribution of these three factors during each phase of the latency/reactivation cycle will be reviewed.

Introduction 21 ____________________________________________________________________________________________________

1.4.2. Establishment of latency The human alphaherpesvirus HSV-1 is generally present in the population. Approximately 90 % of adults are latently infected (Mitchell et al, 2003). Several studies have examined how much HSV-1 is present in infected individuals. Detection of HSV-DNA by PCR in single human TG neurons obtained by a method of cell separation or by laser capture microscopy from authopsized persons without signs of acute HSV infection showed that respectively 3% and 2 to 10% of the single neurons contained latent HSV-DNA (Cai et al, 2002; Wang et al, 2005). These studies also showed that specifically the neurons and not satellite or supportive cells in the TG contained latent virus. PCR analysis of single TG neurons of latently infected mice, obtained after perfusion of mice with Streck’s tissue fixative, a technique called contextual analysis of latency, showed that 1.9 to 24% of these neurons were HSV-DNA positive (Sawtell, 1997). The number of infected neurons in mice correlated with the viral inoculum size and the surface size treated with virus. No such correlation was found after HSV infection of rabbits (Thompson & Sawtell, 2000b; O’Neil et al, 2004).

Figure 5. Schematic representation of an alphaherpesvirus infection of TG neurons. Entry of a virus particle in a cell of the epithelial mucosa (A) is followed by spread to new epithelial cells and formation of new infectious virus particles (B). Progeny virus can enter the axon endings (C) and is transported in the axon via retrograde axonal transport to the neuronal cell body (D) where a productive infection is initiated or a latent infection is installed (E). The decisive factors that lead to a latent infection instead of a productive infection, and that are thought to be based on an interplay between the virus, the neuron, and the immune system, are poorly understood.

22 Chapter 1 ____________________________________________________________________________________________________

The general presence of latent HSV-1 in the population and the high latent viral DNA burden in the ganglia is in big contrast with the high replication rate and extensive spread of alphaherpesviruses in non-neuronal cells. These properties of viral replication in cell cultures show that alphaherpesviruses are excellently designed for lytic infections. The fact that latent infections appear to be installed efficiently in neurons is indicative for a balance that must exist between the repressive environment created by the neurons and the immune system and the viral properties to promote lytic infection (Figure 5). In the next paragraphs, properties of each factor that contribute to the final decision between a latent or lytic infection upon infection will be described. 1.4.2.1. Neuronal properties creating a favorable environment for establishment of latency Neurons are the predominant cell type in which latent alphaherpesvirus infections are induced and therefore must have properties that differentiate them from other cell types and create an environment favorable for latent infections. Several properties of neurons have been proposed to serve to this end. A productive alphaherpesvirus infection initiates by the formation of a complex between the viral tegument protein VP16 and cellular proteins HCF and Oct-1, followed by binding of this complex to IE promoter sequences, resulting in their activation. In neurons however, the formation of this transcription initiation complex is hampered in several ways. First of all, it has been hypothesized that VP16 does not reach the nucleus in sufficient amounts to efficiently start a productive infection, seen the long distance this protein has to travel to reach the nucleus (Roizman & Sears, 1987; Kristie & Roizman, 1988). Furthermore, in contrast to other cell types, HCF has been shown to be localized mainly in the cytoplasm of neurons which hinders the interaction with VP16 (Kristie et al, 1999). Oct-1 in turn is low abundant in neurons and other factors of the Oct-family do not interact with VP16 thereby minimizing the formation of a functional transcription initiation complex (He et al, 1989). Besides the problems in starting viral transcription by the lack of a good initiation complex, IE protein expression has been shown to be differentially regulated in neurons by neuro-specific factors compared to other cell types. For example, in transgenic mice containing the ICP4 promoter, this promoter is activated in Schwann cells of latently infected mice but not in TG neurons (Taus & Mitchell, 2001). The ICP0 promoter is differently regulated in TG neurons of transgene mice depending on the age and activation seems to be associated with binding of the neuronal transcription factor Olf-1 (Devireddy & Jones, 2000). During lytic infection,

Introduction 23 ____________________________________________________________________________________________________

ICP0 has been shown to target ND10 components for proteasome dependent degradation. Several studies report however that neuron-like cells contain abnormal ND10 structures or that neurons lack ND10 (Lam et al, 1995; Hsu & Everett, 2001; Negorev & Maul, 2001). Therefore, the disturbed interaction with ND10 may lead to inefficient activation of the replication cycle (Jones, 2003). Another neuron-specific family of transcription factors, Brn3, has been shown to have the potential to regulate IE expression in neurons (Hagmann et al, 1995). The neuronal property to produce arginase and the associated lack of arginine has been thought to restrict viral replication since arginine has been shown to be necessary during virus replication (Becker et al, 1978; Wildy et al, 1982; Yu et al, 2001). A very important neuronal characteristic is the fact that these cells are terminally differentiated and therefore contain only low levels of proteins associated with DNA replication and in this way suppress lytic viral infection (Nichol et al, 1996). Neurons have also been shown to be inefficient at DNA repair (Gobbel et al, 1998; Nouspikel & Hanawalt, 2000) while DNA damage proteins are important during HSV replication (Lilley et al, 2005). Another neuronal property advantageous for induction of a latent infection is that neurons normally do not express MHC-I complexes so that the virus can not be presented to CD8+-T cells (Wong et al, 1985). Infection however triggers the neurons to produce MHC-I (Pereira et al, 1994; Pereira & Simmons, 1999) but this will be discussed in a next section. Finally, it is hypothesized that certain subtypes of neurons have different protein expression patterns and are therefore more suited for containing latent HSV-1 than others. Margolis et al (2007) observed that neurons positive for a specific oligosaccharide antigen detected by the monoclonal marker A5 are preferentially latently infected, while KH10 positive neurons seem to support a productive infection. This observation will be discussed later. No correlation could be found beween latency and other parameters of neurons such as neuron size, absence or presence of CGRP and substance P or other cell surface expressed glycoconjugates, oligosaccharides or lectins recognized by monoclonal antibodies SSEA3, LD2, BSIL4 and LA4 (LaVail et al, 1991; Margolis et al, 1992; Yang et al, 2000; Margolis et al, 2007). All these factors provide possible explanations for the fact that neurons are the predominant cell type in which latent infections are installed. Several in vitro studies show however that alphaherpesvirus infection of neuronal cultures results in productive infection of all neurons (Wigdahl et al, 1984; Wilcox & Johnson, 1988; Geenen et al, 2005 and 2007). Impairment of the immune system of mice also results in productive virus infection in neurons (this will be discussed in the following section). This indicates that neuronal properties alone are not sufficient to direct incoming alphaherpesviruses in a latent state.

24 Chapter 1 ____________________________________________________________________________________________________

1.4.2.2. Role of the immunity during establishment of latency 1.4.2.2.1. Presence of immune cells and inflammatory cytokines in trigeminal ganglia during establishment of latency Immediately upon infection at the periphery, the virus is subject to the innate immune system. Important early defense mechanisms are the production of IFN−α and –β, the lysis of virus infected cells via the alternative/lectin-binding complement pathway (in absence of antibodies) and the induction of cellular cytotoxicity via activation of macrophages, neutrophils and natural killer cells (Wittmann et al, 1980; Martin & Wardley, 1984; Pol, 1990; Kimman et al, 1992; Nauwynck & Pensaert, 1995a). Virus particles that are not removed by this aspecific early immune response enter the axon endings and are transported to neurons in the ganglia. Several studies with in vivo mice models showed that also in the ganglia, the virus is subject to the immune system. After inoculation of mice with HSV-1 at the cornea, HSV-1 antigens are detected in the TG already at 2 dpi, reaching peak titers around day 3 to 5 and declining between day 7 to 10. After 10 dpi, HSV-1 antigens can no longer be found in the TG, indicating that a uniform latent infection has been installed at that timepoint (Shimeld et al, 1995; Liu et al, 1996; Kodukula et al, 1999). The decline in HSV-1 antigens detected in the TG strongly coincides with an infiltration of immune cells and production of cytokines in the TG. Liu et al (1996) reported an infiltration of macrophages already at 3 dpi and γδ-T cells at 5 dpi which were very closely associated with neurons. CD4+ and CD8+ cells were found to infiltrate the TG around day 5 to 6 after infection (Cantin et al, 1995; Lang & Nikolich-Zugich, 2005) with a strong accumulation of CD8+-T cells between 7 to 12 dpi (Liu et al, 1996). NK cells also infiltrated early during infection (Liu et al, 1996). Increased quantities of iNOS (nitric oxide synthetase), TNF−α (tumor necrosis factor alpha) and IFN−γ mRNA were already detected at 3 dpi by Kodukula et al (1999) via semi-quantitative RT-PCR assays. These authors also showed that macrophages are the main source of NO and TNF−α in the TG and γδ-T cells are important IFN−γ producers. They also detected IL-12 mRNA from 5 dpi. An increase in cells producing IL-4 was found after immunohistochemical examination between 3 and 7 dpi and IL-4 producing cells differed from IFN−γ producing cells (Liu et al, 1996). Another study based on immunohistochemistry detected IL-2 and IL-4 positive cells only after 10 to 14 dpi, while they never could detect IL10 in the TG (Shimeld et al, 1997). At day 3 pi however, they detected high numbers of IL-6 and/or TNF−α positive cells and low numbers of IFN−γ positive cells surrounding HSV

Introduction 25 ____________________________________________________________________________________________________

antigen positive neurons in the TG. Using RT-PCR, Halford et al (1997) detected IL-2, TNF−α, IFN−γ and IL10 mRNA at 7 dpi. Using ELISA, they detected IL-2, IL-6, IL-10 and IFN−γ in TG homogenates also at 7 dpi while IL-12 could not be detected in the same ELISA setup. IFN−α mRNA was detected by RT-PCR at 6 dpi in TG of mice infected with WT-HSV (Peng et al, 2005), while IFN−α was detected in the TG via an IFN bio-assay at 3 days post infection (Carr et al, 1998). In summary, the following cytokines have already been detected at the protein level in the TG during the period in which HSV-1 establishes a latent infection in mice: IFN−α/β, IFN−γ, TNF−α, IL-2, IL-4, IL-6, IL10 and also NO producing enzymes. Several studies have shown the importance of the immune cell infiltration and the associated production of cytokines in the establishment of a latent alphaherpesvirus infection. Knock-out mice in γδ-T cells, mice treated with monoclonal antibodies directed against γδ-T cells or mice chemically depleted in macrophages all showed higher viral titers in the TG and higher mortality related to encephalitis after infection than WT-mice, indicating the involvement of these cell types in the establishment of latency (Kodukula et al, 1999; Sciammas et al, 1997). Also knock-out mice in IFN−γ (Minami et al, 2002), TNF−α (Minami et al, 2002) and IFN−α/β receptor (Leib et al, 1999; Halford et al, 2006) showed higher virus titers in the TG after infection than WT-mice. However, one study using knock-out mice in IFN−γ or the IFN−γ receptor reported that there was no significant difference in TG viral titers or the timing of establishment of latency (Cantin et al, 1999), indicating that the role of IFN−γ might not really lie in the establishment of latency but rather in retaining the virus in the suppressed state. Higher viral titers were also observed when either IFN−α, IFN−β (Halford et al, 1997), IFN−γ or TNF−α (Kodukula et al, 1999) were neutralised using antibodies directed to each of these cytokines. In another experimental setup using transgene mice expressing the IFN−α gene under control of an astrocyte specific promoter, it was shown that high IFN−α expression in the TG resulted in highly reduced viral titers in the TG at 3 and 6 dpi and correlated with a reduction in the IE gene ICP27 mRNA expression (Carr et al, 1998). Placing IL-6 under control of this promoter also resulted in significantly reduced virus titers in the TG at day 6 pi but not at day 3 pi, and also here, the correlation with reduced amounts of ICP27 mRNA was found (Carr & Campbell, 1999). By injecting mice with aminoguanidine, an inhibitor of NO production, it was shown that NO depletion also resulted in a higher viral load in the TG. All these studies show the importance of different immune cells and cytokines of the innate immune system present in the TG of mice after infection for the establishment of latency.

26 Chapter 1 ____________________________________________________________________________________________________

Some studies however indicate that the adaptive immune system may also be of importance in establishing latency in neurons. In TCRα-knock-out mice which could not produce αβ-T cells, which are part of the adaptive immune system, replicating virus persisted in a small number of neurons and these mice ultimately died of encephalitis (Sciammas et al, 1997). In SCID mice which lack an adaptive immune response but exhibit strong innate immunity, virus replication was controlled and latency established in some but not in all neurons (Gesser et al, 1994; Ellison et al, 2000). Transfer of αβ-T cells restored the ability of SCID mice to completely restrict HSV-1 replication in the TG and establish uniform latency (Minagawa & Yanagi, 2000). As mentioned before, the infiltration of virus specific CD8+-T cells is described from around day 5 to 7 pi. Several authors hypothesized that this is too late to really play a significant role in the establishment of a latent infection (Liu et al, 1996; Lang & Nikolich-Zugich, 2005). Mice depleted in CD8+-T cells however were not able to completely clear virus from sensory ganglia and virus did spread to the brain, causing encephalitis (Simmons & Tscharke, 1992; Lang & Nikolich-Zugich, 2005). CD4+-T cells have also been shown to infiltrate in the TG but their role in HSV-1 latency has not yet been explored (Khanna et al, 2004a; Decman et al, 2005a). Summarizing, the discussed data indicate that the innate immune system and especially cytokines play an important role during the establishment of latency. It is less clear if the adaptive immune system is involved in efficiently establishing a uniform latent infection or if it rather is involved in maintaining virus in the repressed state after latency has been establised. 1.4.2.2.2. Interferons and other inflammatory cytokines: mode of action In a previous paragraph, it was described that IFNs are present in the TG at the time that a latent infection is installed. Therefore, this section will summarize the antiviral effects against HSV-1 and PRV induced by IFNs, as well as the mode of action of other cytokines present in the TG at the time of latency establishment. The production of IFN is one of the best documented antiviral responses of cells. IFNs induce an antiviral state in cells by binding to their receptors present on the plasma membrane. Use of micro-arrays showed that upon binding of IFN to its receptor, several hundred genes are transcriptionally regulated (de Veer et al, 2001). Three families of such IFN-induced genes have been studied extensively with respect to their antiviral activities, but it is clear that there must exist many more antiviral pathways induced by IFN than those discovered up to now.

Introduction 27 ____________________________________________________________________________________________________

The best studied IFN induced genes encode double-stranded RNA-activated protein kinase (PKR), the 2’,5’-oligoadenylate synthetase (OAS) and the Mx proteins (reviewed by Levy and Garcia-Sastre, 2001). PKR is a cellular protein that becomes activated upon binding to dsRNA and subsequently phosphorylates and thereby inactivates the eukaryotic translation initiation factor eIF-2α. This inactivation leads to a general inhibition in protein synthesis and thereby inhibits viral protein expression and replication in virus infected cells. OAS becomes activated by dsRNA and forms 2’,5’-oligoadenylate which binds to and activates RNaseL. Subsequently, RNaseL cleaves mRNA and rRNA leading to an inhibition of protein expression. The mechanism of action of Mx proteins is not yet completely understood. Studies in mice embryo fibroblasts from WT or PKR or RNaseL knock-out mice treated with IFN−α showed that both PKR and RNaseL have a suppressive effect on HSV-1 replication (Khabar et al, 2000). Both pathways also suppressed HSV-1 replication in primary TG cultures in the presence of IFN−β (Al-khatib et al, 2003; Carr et al, 2003). The antiviral effect of IFN−γ on HSV-1 infection in TG cultures does not seem to be dependent on one of these mechanisms (Austin et al, 2006). IFNs also induce antiviral effects by other mechanisms than those related to the three families of IFN-induced genes described above. IFN−α, −β and −γ have also been shown to upregulate Sp100 and PML proteins, both components of ND10 (Chelbi-Alix et al, 1995; Grotzinger et al, 1996) which may be a part of the anti-HSV-1 state induced by IFNs (Chee et al, 2003). IFN−γ also decreases cdk2 and cdk4 activity by inducing cdk inhibitors which are necessary for HSV-1 replication and α, β and γ gene expression (Mandal et al, 1998; Schang et al, 1998 and 2002). IFN-stimulated gene 15, that is produced after IFN stimulation, has recently been shown to also display anti-HSV-1 properties (Lenschow et al, 2007). Several studies document that both type I (α/β) and type II (γ) IFNs can block HSV-1 infection at the level of IE gene transcription in primary mouse macrophages and in human epithelial cells and lung fibroblasts, via an unknown mechanism (Mittnacht et al, 1988; Oberman & Panet, 1988 and 1989, De Stasio & Taylor, 1990, Nicholl & Preston, 1996). Pierce et al (2005) showed that IFN−β and –γ could reduce ICP0 expression to undetectable levels in 70% of infected Vero cells and provided an uncharacterised block at the level of viral DNA synthesis in the other 30% of infected cells. It was shown by Tonomura et al (1996) that IFN−α also reduced IE180 gene expression of PRV in Vero cells. Presumably, more antiviral effects of IFN await discovery.

28 Chapter 1 ____________________________________________________________________________________________________

Besides the direct antiviral activities, IFNs can also stimulate effector functions of NK cells, CTLs and macrophages, upregulate expression of MHC-I and -II molecules, induce antibody synthesis by B cells and stimulate the proliferation of memory-phenotype T cells, in this way modulating the innate and adaptive immune system (Guidotti & Chisari, 2001). Very little is known about the possible role of the other cytokines and NO in the establishment of latency. Not much is known on direct antiviral mechanisms activated by TNF−α (Herbein & O’Brien, 2000). They seem to comprise both apoptotic and non-apoptotic signaling pathways, but the major action seems to be to synergize with IFN−γ (Benedict, 2003). Direct antiviral effects of NO on HSV-1 have been reported in several cell lines in vitro (Croen et al, 1993; Karupiah & Harris, 1995). Interleukins in turn do not have direct antiviral functions, but are rather indirect immunoregulators (Janeway et al, 2001). In summary, IFNs and other cytokines appear important in the suppression of HSV-1 replication but it is unclear whether cytokines alone are sufficient to drive HSV in a latent state of infection. 1.4.2.2.3. Alphaherpesvirus interference with the interferon-induced antiviral state In the previous section, it was described that production of IFNs is one of the strongest antiviral mechanisms of infected cells and it appears to be an important player in preventing lytic virus infection. During

the long co-evolution

with

their hosts however,

alphaherpesviruses have developed several mechanisms to counteract the intracellular antiviral state induced by interferon. ICP0 is an IE protein of HSV-1 that displays important anti-IFN capacities (Harle et al, 2002). Recently, it has been shown to antagonize the STAT-1 dependent host response (Halford et al, 2006) and both HSV-1 ICP0 and the BoHV-1 ICP0 orthologue have been shown to either directly or indirectly target interferon response factor 3 (IRF3) to proteasome dependent degradation (Lin et al, 2004; Melroe et al, 2007; Saira et al, 2007), thereby inhibiting transcription of genes promoted by this transcription factor, including IFN−β production. It also prevents rRNA degradation independent of the classical RNaseL antiviral pathway indicating the existence of another RNase that is part of the host antiviral response to viral infection (Sobol & Mossman, 2006). The ICP0 orthologue of PRV, EP0, was also shown to be important to overcome the IFN mediated antiviral response in cells of its natural host (Brukman & Enquist, 2006). Two more properties of ICP0 are known to counteract specific anti-HSV actions of interferons that were described in previous sections. ICP0 is known to be

Introduction 29 ____________________________________________________________________________________________________

a promiscuous transactivator because it induces the expression of HSV α, β and γ genes as well as cellular proteins. It is considered to be a multifunctional protein directed primarily towards modification of cellular functions to promote viral gene expression and viral productive infection (Roizman & Knipe, 2001). An important effect of ICP0 on cellular proteins is the de-repressive effect on infection by targeting PML of ND10 for destruction by proteasomes, thereby disrupting ND10 and promoting lytic infection (Everett et al, 2000). Besides ICP0, HSV is known to express two late proteins that help to overcome the IFN induced antiviral state. ICP34.5 recruits a cellular phosphatase to dephosphorylate eIF-2α, thereby releasing the translational block induced by PKR (He et al, 1997). US11 in turn, was found to bind to and inhibit PKR and was also recently shown to inhibit OAS (Cassady et al, 1998; Sanchez & More, 2007). A delicate balance between the environment favorable for latent infections created by the neurons and the immune system and the virus promoting lytic infection is thought to decide the outcome of infection. Because ICP0 appears to be the most important viral counteraction against antiviral effects induced by IFNs, some authors speculate that this balance between a lytic and latent infection can even be reduced to the net effect of the cross regulation between IFN and ICP0. They suggest a model in which interferons block functions of ICP0 that are required for the start of a lytic infection. In this model, establishment of latency comes down to the delivery of the viral genome to the nucleus and an IFN induced block in IE gene transcription or IE protein expression (Efstathiou & Preston, 2005). 1.4.2.3. Alphaherpesvirus properties promoting latency Alphaherpesviruses like HSV-1, PRV and BoHV-1 encode only one type of transcript that is believed to play a role in viral latency, the latency associated transcripts (LATs). Many studies show that the virus genome is repressed during latency and transcription is restricted to the production of these RNA species (Roizman & Knipe, 2001). LATs were first discovered by ISH in 1984 (Stroop et al, 1984) and detected by northern blotting in 1987 (Spivack & Fraser, 1987; Stevens et al, 1988). The LAT region is located to the viral repeat sequences flanking the UL region of the virus genome. Multiple transcripts are transcribed from the LAT region, including the 8.3 kb primary transcript that is transcribed antisense to the ICP0 gene and 2 stable introns of 2.0 and 1.5 kbp. The 2.0 kb LAT intron is the most studied one because it is the only abundant HSV transcript found during latency. Further splicing of the 2.0 kb LAT RNA occurs within neuronal cells to produce an additional stable

30 Chapter 1 ____________________________________________________________________________________________________

RNA species of 1.5 kb (Zwaagstra et al, 1990; Farrell et al, 1991). The 2.0 kb LAT is also produced late during lytic infection but to a lesser extent than in a latent infection. This is caused by the repressive effect of ICP4 binding to the LAT promoter on the LAT expression during lytic infection (Spivack & Fraser, 1988). During acute infection, LAT can promote the lytic infection by slowing down the IFN production (Peng et al, 2005). Because LATs are the only abundant viral transcript found during a latent infection of these viruses, they are often seen as a hallmark for latent infection and many studies are dedicated to find possible functions for this transcript. Nevertheless, several lines of evidence indicate that the role of LATs may not be as widespread as initially thought. First of all, it has been shown that LATs are not essential for any step of the latency/reactivation cycle (Javier et al, 1988; Steiner et al, 1989). Secondly, it has been shown in several independent studies that there are far more neurons that contain HSV-DNA than there are LAT positive neurons, indicating that one has to be careful by using LATs as a setpoint to state that a neuron is latently infected. The use of in situ PCR and RT-PCR to detect viral DNA and ISH to detect LATs showed that 67 to 95% of the HSV-DNA+ neurons did not show LAT expression in mice and rats (Ramakrishnan et al, 1994 and 1996; Mehta et al, 1995, Maggioncalda et al, 1996). A recent study using the same techniques revealed that also in human TG neurons, 80 to 98% of the HSV-DNA+ neurons were LAT- (Wang et al, 2005). Some argue that ISH is not sensitive enough to detect the LATs and that this explains the big discrepancy between DNA+ and LAT- neurons. In this context, a study that used in situ RT-PCR to detect LATs found an equivalent number of LAT+ and DNA+ neurons. However, in this study, it could not be ruled out that the virus was no longer in a latent state (Ramakrishnan et al, 1996). To find an explanation why LATs appear to be expressed in some neurons and not in others, Chen et al (2002b) looked if there was a correlation between the HSV genome copy number in TG neurons and the expression of LATs. No clear correlation could be found and they suggested that neuron-specific factors may play a role in LAT expression during latency. In this context, it is interesting that a recent study reported that HSV latency is installed preferably in a neuronal subset of mice TG neurons that is characterised by the expression of a polysaccharide antigen recognized by the monoclonal antibody A5 and that LATs regulate the induction of latency in this subtype (Margolis et al, 2007). However, these authors used LAT expression as a hallmark for latently infected neurons, suggesting that it might be more appropriate to interpret these results as that A5+ neurons favor the expression of LAT RNA during latent infection compared to other cell types. Despite these remarks, many studies are

Introduction 31 ____________________________________________________________________________________________________

undertaken to find out in which way LATs are important for a latent infecton. In the next paragraphs, the effects attributed to LATs will be summarized. The most important phenotype of LAT- mutants in rabbits is the reduced reactivation capacity (Trousdale et al, 1991; Bloom et al, 1994; Perng et al, 1994 and 1996). In mice however, this reduced reactivation seems to be virus- and mouse strain specific (Javier et al, 1988; Deshmane et al, 1989; Ho & Mocarski, 1989; Cook et al, 1991; Sawtell & Thompson, 1992; Block et al, 1993; Devi-Rao et al, 1994; Perng et al, 2001) but transgenic mice expressing LATs could augment reactivation of LAT- mutants to a level comparable to wild type mice (Mador et al, 2003). One possibility is that the reduced reactivation phenotype may be explained by a reduced efficiency of LAT- mutants to establish a latent infection. This is supported by the observation that there are less latently and more productively infected neurons after infection with LAT- mutants compared to WT virus (Sawtell & Thompson, 1992; Thompson & Sawtell, 1997). This has been attributed to the fact that i) after infection with LAT- mutants, the normal repression of LATs on productive cycle genes is not present leading to neurons that are destroyed by a lytic infection (Chen et al, 1997; Garber et al, 1997) and ii) LAT- mutants lack the anti-apoptotic effect attributed to LATs resulting in more infected neurons undergoing apoptosis and less latent neurons remaining and iii) recent studies suggest that LATs might also influence survival of neurons by interfering with cellular protein production. Interestingly, the idea that LATs promote a higher survival of neurons is also supported by the finding that IFNs, which induce an antiviral state in cells and signal via STAT-1, upregulate HSV-1 LAT expression by binding of STAT-1 to the LAT promoter (Kriesel et al, 2004). Several mechanisms are addressed to LATs to explain how LATs normally suppress the production of productive cycle genes. One of the most appealing ones is that LATs may act by an antisense mechanism to silence ICP0 gene expression, the transactivator of lytic infections. This is supported by the observation that the expression of LATs in trans could inhibit transactivation of gene expression by ICP0 (Farrell et al, 1991) and that this suppressive effect was not due to a LAT effect on the ICP0 promoter (Mador et al, 1998). Other studies challenged this hypothesis by showing that infection with LAT- mutants did not result in increased levels of ICP0 transcripts in mice ganglia (Chen et al, 2002a) and that LAT expression in trans had no effect on ICP0 mRNA expression, stability, accumulation, splicing and translation in non-neuronal cells (Burton et al, 2003). Two other studies state that LATs do not work antisense to ICP0 but are able to regulate ICP0 at a post-transcriptional level. They show that in mice TG, LATs favor the accumulation of unspliced, intron containing,

32 Chapter 1 ____________________________________________________________________________________________________

inactive ICP0 over the spliced, active ICP0 form (Chen et al, 2002a; Maillet et al, 2006). Another possible mechanism by which LATs could repress productive cycle protein expression is by interacting with ribosomes. It was shown that LATs may play a structural role in ribosomal complexes and thereby affect the functioning of the translational machinery and protein production (Goldenberg et al, 1997; Ahmed & Fraser, 2001). Recently, it has been shown that LATs can manipulate the cellular histone modification machinery to increase the amount of heterochromatin on lytic gene promoters during the course of establishment of latency, thereby repressing these promoters (Wang et al, 2005). Besides the repressive action of LATs on the induction of lytic gene and protein expression, LATs also seem to have an anti-apoptotic function. During lytic infection, several viral proteins of HSV-1 and their orthologues in other alphaherpesviruses have been shown to have an anti-apoptotic function, for example ICP27, ICP22, US3, US5 and ICP4, allowing the production of new virus particles by postponing the apoptosis of the infected cells (Aubert & Blaho, 2001). Since during a latent infection, none of these proteins are produced, a possible function for LATs as being anti-apoptotic has been investigated. Several studies addressed anti-apoptotic properties to HSV-LAT in vivo in neurons of mice and rabbits and in vitro in different cell types (Perng et al, 2000 and 2002; Inman et al, 2001; Ahmed et al, 2002; Jin et al, 2003; Branco & Fraser, 2005). The LAT orthologue of BoHV-1, the LR gene, has also been shown to inhibit apoptosis (Ciacci-Zanella et al, 1999). The notion that LATs have an anti-apoptotic function has been challenged by Thompson & Sawtell (2000a and 2001). By performing TUNEL stainings on TG of mice infected with WT and LAT- HSV, they found that the higher survival of neurons infected with WT virus was not due to an anti-apoptotic function of LAT, but rather to the suppressive effect on lytic gene expression that promoted neuron survival. Kent et al (2003) hypothesized that these observed differences may be due to the differences in animal models employed. Recent studies have suggested several possibilities how LATs could exercise their anti-apoptotic function. Transient transfections of LATs in neuro-2A cells could inhibit caspase 8 and 9 maturation, thereby inhibiting their role in the induction of apoptosis (Henderson et al, 2002; Jin et al, 2003). The LR gene of BoHV-1 was found to be able to suppress caspase 3 and 9 proteolytic activation (Henderson et al, 2004), inhibiting apoptosis in this way. Another possible mode of action by which LAT inhibit apoptosis is by the upregulation of the anti-apoptotic protein Bcl-x(L) in favor of the pro-apoptic protein Bcl-x(S) in neuro-2A cells (Peng et al, 2003). It was also recently reported that a miRNA encoded by the LAT gene exhibited an anti-apoptotic function by modulating

Introduction 33 ____________________________________________________________________________________________________

the TGF-β signaling pathway, a known inducer of apoptosis (Derynck et al, 2001; Gupta et al, 2006). Besides the probability that LATs increase the neuronal survival by suppressing lytic viral gene transcription or by interfering with proteins that are known to direct cells in apoptosis, some recent studies showed that LATs can also interfere with cellular protein expression levels and augment the survival of neurons in this way. Hamza et al (2007) showed that LAT transfection in primary rat embryonic TG neurons decreased the level of CGRP which might reduce the neuro-inflammatory response and in this way protect the host neuronal cells during latency. It was also shown that LATs could stimulate the expression of heat shock proteins in human neuroblastoma cells which led to protection of these cells from stress (Atanasiu et al, 2006). A few studies suggest a function for LAT directly related to the increase of reactivation and not to a higher survival of neurons. Although it is generally believed that LATs are present as RNA in the cell and do not lead to protein production, one group suggested the presence of a protein encoded from an open reading frame of the 2.0 kb locus. This protein has been thought to act somewhat similarly to ICP0, in this way being able to substitute for ICP0 during reactivation and initiate lytic gene expression from latent viral genomes (Thomas et al, 1999 and 2002). Naito et al (2005) also identified a protein encoded by the LAT promoter region but the function of this protein still has to be determined. Besides the possible role of proteins encoded by LATs in initiating reactivation, it was also shown that the CRE sequence in the LAT promoter was essential to induce reactivation of HSV by hyperthermic stress in latently infected mice TG neurons. LAT- mutants had no effect on the genome copy number and the amount of latent virus present in these neurons (Marquart et al, 2001). 1.4.2.4. Does virus replication preceed latency establishment? Quantitative real time PCR studies on single neurons obtained by the same methods as described to quantify numbers of infected neurons in section 1.4.2 showed that the genome copy number per neuron may vary between 10 and 1000 in mice and had a mean copy number per neuron of respectively 50 and 11,3 in humans with high variations (Sawtell, 1997; Cai et al, 2002; Wang et al, 2005). The high variation in viral genome copy number per infected neuron raises questions about the possibility of intracellular viral replication before the induction of latency. Infection of mice with virus mutants defective in IE protein production, e.g. ICP4- and ICP0- mutants, led

34 Chapter 1 ____________________________________________________________________________________________________

to succesful establishment of a latent infection of these viruses, showing that IE gene expression and subsequent DNA replication are not required to establish latency (Preston and Nicholl, 1997; Samaniego et al, 1998; Marshall et al, 2000). Other studies using thymidine kinase (TK) negative mutants that show impaired DNA replication reported that high virus genome copy numbers (over 100 copies) could still be present in single neurons, suggesting that high copy numbers of viral genomes during latency may be derived from multiple virions that have entered the same neuron (Thompson & Sawtell, 2000b). These data indicate that viral replication is not required to induce a latent infection. The very high viral genome copy numbers found in certain neurons however makes it difficult to exclude that latency may also be installed after primary viral DNA replication was initiated. 1.4.3. Maintenance of latency Once the alphaherpesvirus is brought into a repressed state during the establishment of latency, the virus can remain in this state for a long time without the production of new virus particles. This phase is called the maintenance of latency. Again, the interaction between virus, neurons and immune system plays a crucial role, with the immune system in a leading role. 1.4.3.1. Continuous presence of components of the adaptive immune response in TG during maintenance of latency As described in the previous section, the disappearance of HSV-1 antigens in the TG coincides with the infiltration of immune cells and cytokine production in the TG, leading to the establishment of a uniform latent infection around day 7 to 10 after infection. Several studies however indicate that this infiltration of immune cells of the innate and adaptive immune response and associated cytokine production lasts for a much longer time period than that needed for latency establishment. Infiltrated CD4+-T cells, virus specific and non-virus specific activated CD8+-T cells (Khanna et al, 2003; Van Lint et al, 2005) and macrophages were found in infected TG of mice for periods from 40 dpi up to 92 dpi while they were not detected in ganglia of uninfected mice (Shimeld et al, 1995; Liu et al, 1996; Khanna et al, 2003). In TG from latently infected humans, infiltrated macrophages and CD8+-T cells were also found (Theil et al, 2003; Verjans et al, 2007). RT-PCR analysis of TG of infected mice revealed the presence of mRNA encoding the cytokines IFN−γ, TNF−α, IL-2, IL-10 and the

Introduction 35 ____________________________________________________________________________________________________

T-cell attractant chemokine RANTES for periods up to 135 dpi (Cantin et al, 1995; Halford et al, 1996; Carr et al, 1998). Another recent RT-PCR based study showed the presence of chemokines like macrophage inflammatory protein 1-a (MIP-1a), MIP1-b, MCP-1 and RANTES and their receptors CCR1, CCR2, CCR3 and CCR5 at 30 dpi in mice TG (Cook et al, 2004). Using immunohistochemistry, cells positive for IFN−γ, TNF−α, IL-2, IL-4, IL-6 and IL-10 could be detected at 30 dpi, some even up to 92 dpi. The number of positive cells for each cytokine at that timepoint was reduced in comparison to peak numbers of cells observed between 10 and 14 dpi but was still consistently higher than observed numbers in TG of uninfected mice. Only the number of TNF−α expressing cells seemed to remain at the same high elevated number (Liu et al, 1996; Shimeld et al, 1997). The presence of IFN−γ, TNF−α and RANTES was also found in latently infected human TG (Theil et al, 2003; Hufner et al, 2006). Besides immune cells, cytokines and chemokines, high anti-HSV-1 serum antibody levels were also detected up till 125 dpi (Halford et al, 1996). HSV specific plasma cells were detected in spinal cords and lumboscral ganglia of mice and guinea pigs up till 10 months after intra-vaginal HSV-2 infection (Milligan et al, 2005). The presence of different parts of the adaptive immune system in the TG at time points far past the induction of latency leads to the hypothesis that the acquired immune system plays an important role in the maintenance of latency. The long retention of CD8+-T cells and immune cells in general in the ganglia is however contradictory to the statement that the transcription of viral genes during latency is restricted to the LATs. However, an increasing amount of studies report the presence of low levels of viral proteins produced during the latent phase of infection in some neurons without the production of new virus particles. ICP4, ICP0 and TK mRNA was detected by RT-PCR and ISH in unrelated studies in TG of infected mice at time points when a latent infection should be installed (>30 dpi) (Kramer & Coen, 1995; Chen et al, 2002a; Feldman et al, 2002). ICP4 was also detected at the protein level in TG of infected rabbits at 200 dpi (Green et al, 1981). Even late viral protein mRNA of gC was detected by ISH in about 1 neuron per 10 TG of infected mice (Feldman et al, 2002). Several lines of indirect evidence also point in the direction of incidence of viral gene transcription in latently infected neurons: i) the infiltrated CD8+-T cells are specifically retained around neurons, the cell type that is thought to harbor the latent virus in the TG (Cantin et al, 1995; Liu et al, 1996; Khanna et al, 2003) and ii) infection with a LAT- mutant did not result in reduced cytokine expression levels showing that LATs are not the trigger for this process (Carr et al, 1998; Chen et al, 2000). Another apparent contradictory element for the long retention of

36 Chapter 1 ____________________________________________________________________________________________________

CD8+-T cells in ganglia is the fact that neurons have been described to be a cell type that does not express MHC-I (Wong et al, 1985). Studies by Pereira et al (1994 and 1999) demonstrated however that sensory neurons do express MHC-I molecules during an acute HSV-1 infection. This could explain how viral antigens can be presented to the infiltrating CD8+-T cells. This MHC-I expression was however no longer detectable once the latent infection was installed. An explanation for this discrepancy could be found in a recent study that showed that CD8+-T cell stimulation only requires very low levels of antigen expression (Purbhoo et al, 2004), leading to the hypothesis that during initiation of reactivation, MHC-I molecules may be produced and present on the cell surface in quantities that are too low to detect but sufficient to present newly produced viral antigens to CD8+-T cells. In summary, latency in ganglia appears not to be a stable, uniform state. The continuously elevated cytokine levels and the retention of immune cells point in the direction of the occurence of frequent initial steps of reactivation. 1.4.3.2. Role of CD8+-T cells during maintenance of latency Based on the data described in the previous paragraph, it appears possible that the latent stage of infection is not an entirely quiescent, stable stage and that virus frequently reactivates in a minority of infected neurons but that the surveilling immune system is able to suppress these early reactivation events. Several studies show an important role for CD8+-T cells in this suppression. A first indication for the importance of CD8+- T cells was the high infiltration and retention of CD8+-T cells around neurons in the TG of HSV-1 infected mice in vivo (Cantin et al, 1995; Liu et al, 1996; Khanna et al, 2003) and the observation that more than 60% of these CD8+-T cells were specific for a single immunodominant epitope of the HSV-1 glycoprotein B (Khanna et al, 2003). A second indication for a role of CD8+-T cells in blocking HSV-1 reactivation came from studies using ex vivo cultures of latently infected ganglia. In this model system, ganglia from latently infected mice are removed from anesthesized mice, dissociated and brought in culture in vitro. This process leads to efficient reactivation of virus from latency and detectable viral proteins after 2 days post explant. A study of Liu et al (2000) showed that addition of CD8+-T cells, present in TG of mice 14 days after corneal infection, to such ex vivo cultures could maintain the virus in a repressed state. In additon, it was shown that a CD8+-T cell clone specific for the immunodominant epitope of gB also was able to block reactivation of HSV-1 from latency in a dose dependent MHC-I restricted fashion in ex vivo ganglion cultures (Khanna et al, 2003). The importance of CD8+-

Introduction 37 ____________________________________________________________________________________________________

T cells to control HSV-1 infection was also confirmed in a study showing that CD8+-T cell deficient mice failed to control HSV-1 infection and that by consequence, mice died from encephalitis between 7 and 12 dpi (Lang & Nikolich-Zugich, 2005). This is however in contrast with another study that showed that in mice lacking CD8+-T cells, no significant differences were found in the persistence of HSV-1 in the TG or in reactivation, leading to the hypothesis that CD8+-T cells are not absolutely required to maintain latency (Stuart et al, 2004). Nevertheless, the strong indications, both in vivo and in vitro, that CD8+-T cells were able to maintain alphaherpesvirus latency suggested a non-cytolytic mechanism of suppression of HSV-1 reactivation from latency. For a long time, it was thought that CD8+-T cells, also known as cytotoxic T lymphocytes (CTLs), controlled virus infected cells by killing them via cytolytic activity (Kagi et al, 1994; Kojima et al, 1994). Only more recently, it was observed that upon antigen recognition, CTLs can act by releasing antiviral cytokines, especialy IFN−γ and TNF−α, and that this can account for a non-cytolytic clearance of the virus from the infected cells (Guidotti et al, 1996; Nakamoto et al, 1997; McClary et al, 2000; Guidotti & Chisari, 2001). In agreement with this, it has been shown that the addition of IFN−γ to ex vivo explant cultures of TG of latently infected mice could substitute largely for the addition of CD8+-T cells in maintaining latency: IFN−γ could maintain latency in about 90% of the cultures (Liu et al, 2001; Decman et al, 2005b). Decman et al (2005b) also showed that the blockade of HSV-1 reactivation from latency in neurons by IFN−γ is associated with an inhibition of the expression of the ICP0 gene in most of the neurons confirming an IFN induced block at the IE gene expression level as mentioned before. In neurons in which the virus can overcome this IE block, a second poorly understood inhibitory step seems to be provided by IFN−γ that can stop reactivation of the virus even after the expression of some late viral proteins (Decman et al, 2005b). The role of IFN−γ in the maintenance of latency was also shown in a study with IFN−γ knock-out mice. The rate of reactivation after UVirradiation was significantly higher in these knock-out mice than in control mice (Minami et al, 2002). What determines whether CD8+-T cells clear HSV from an infected neuron via a cytolytic or a non-cytolic fashion is not yet completely understood. One possible mechanism that has been proposed is based on epitope density. When a CTL recognizes a neuron early during reactivation, only few viral antigens will be presented in MHC-I molecules and the CTL will react by releasing antiviral cytokines. In contrast, when the CTL encounters a neuron late in the reactivation process, the viral epitope density will be high and might result

38 Chapter 1 ____________________________________________________________________________________________________

in the induction of the entire lytic program of CTLs, including lytic granule release causing apoptosis of the neuron (Valitutti et al, 1996; Khanna et al, 2004b). An alternative explanation lies in the observation that memory CD8+-T cells loose granzyme B expression, but maintain expression of IFN−γ. When IFN−γ fails to shut-down HSV-1 gene expression, the memory CD8+-T cell becomes stimulated and regains granzyme B expression and its ability to kill the neuron (Kaech et al, 2002). Recently it was reported that activated virus-specific memory CD8+-T cells express the CD94-NK cell receptor subfamily G2a inhibitory molecule which makes them unable to exert cytotoxicity when interacting with Qa-1b expressing targets. Interestingly, many neurons in the latent TG expressed Qa-1b (Suvas et al, 2006). These studies show the existence of regulatory systems which favor the non-cytolytic pathway in order to protect irreplaceable neurons from destruction by the immune system. Only when reactivation can no longer be blocked by cytokine production, the cytotoxic mechanism is activated, resulting in prevention of virus production by killing of the neuron. In addition, TG neurons have been shown to display remarkable resistance against apoptosis induced by different stimuli, possibly making them less sensitive to cytotoxic effects of CTL (Geenen et al, 2005, 2006 and 2007). Anti-apoptotic properties of LATs may also be important in this respect (Perng et al, 2000 and 2002; Inman et al, 2001; Ahmed et al, 2002; Jin et al, 2003; Branco & Fraser, 2005). The observation that IFN−γ could not completely substitute for CD8+-T cell addition to ex vivo explant cultures in maintaining latency could be explained by the different expression of IFN−γ receptors in neuronal subtypes (Vikman et al, 1998) and indicates that other cytokines produced by CD8+-T cells or other immune cells present in the latently infected TG may also play a role in maintaining latency. How CD4+-T cells aid in maintaining latency is not yet clear (Khanna et al, 2004a), but a certain role must be attributed to this cell type because it has been shown that SCID mice, which have no adaptive immune system and fail to completely repress HSV-1 infection, transferred with CD4+-T cells develop latent HSV infection in the TG (Minagawa & Yanagi, 2000). Also overexpression of IL-2 or IL-4 under control of the LAT promoter in recombinant HSV-1 strains could increase survival of mice to 90 à 100% in comparison to only 13% after infection with the parental virus. This high survival was compromised however by the depletion of CD4+- and CD8+-T cells, again showing that the T cell response is important for latency (Ghiasi et al, 2001 and 2002). A possible role for the humoral adaptive immune system in maintenance of latency also has to be taken into account. Elevated HSV-1 specific antibody titers were detected up to 125 dpi (Halford et al, 1996) and

Introduction 39 ____________________________________________________________________________________________________

it has been described that non-neutralising monoclonal antibodies directed to gB and gE can suppress intracellular virus replication in TG neurons of mice (Oakes & Lausch, 1984). 1.4.3.3. Gene silencing during maintenance of latency Another control mechanism to maintain virus in the latent state that recently starts to be appreciated and is independent of the immune system, is genome silencing by epigenetic mechanisms. Examples are DNA methylation or post-translational modification of aminoterminal tails of histones (e.g. methylation, acetylation, phosphorylation) that associate with gene promoters resulting in differential transcripional activity of genes despite their identical DNA sequence. As described in point 1.3.3, during lytic infection, portions of the HSV-1 genome become rapidly associated with histones that are associated with an open chromatin structure correlating with transcription (Kent et al, 2004). Furthermore, it has also been reported that VP16 recruits histone acetyltransferases and chromatin remodelling enzymes to virus IE promoters which may function to exclude histone deposition at actively transcribed IE gene promoters, all to favor IE gene transcription (Herrera & Triezenberg, 2004). It has been shown that during latency, the LAT promoter, which is active during latency, is enriched with acetylated histone H3, while the HSV-1 polymerase gene showed a decreased association with acetylated histones indicating a condensed chromatin structure and gene silencing (kubat et al, 2004ab). Further studies will have to show the extent of such form of silencing during latency and its role in maintenance of latency. 1.4.4. Reactivation from latency The last step in the latency/reactivaton cycle is the reactivation from the latent state of infection. During reactivation, new virus particles are produced in neurons and transported back to the periphery, causing recurrent symptoms (Figure 6). Among those, cold sores and keratitis are the most observed symptoms for HSV-1. Reactivation of alphaherpesviruses in humans is often correlated with physical and psychological stress and immunosuppression. Also in animal models, physical stress as neurectomy, application of chemical stimuli to the skin, UV-irradiation, mild trauma, ocular iontophoresis of epinephrine and transient hyperthermia and psychological stress induced by disruption of social hierarchy has been shown to trigger reactivation (reviewed in Millhouse & Wighdahl, 2000; Sainz et al, 2001). It is not entirely clear how stress causes reactivation of

40 Chapter 1 ____________________________________________________________________________________________________

alphaherpesviruses but it has been shown that stress can elicit release of neurotransmitters and neurohormones which in turn can modulate the host’s immune system, for example T-cell suppression by adrenocorticotropic hormones, increase or decrease in cytokine production by neurohormones, cathecholamines and glucocorticoids, depletion of natural killer cells and others (Sainz et al, 2001). In summary, stress appears to compromise the immune system which was described in the previous section to be of major importance to maintain latency, in this way giving the virus the chance to overcome its repression and start the production of new virus particles.

Figure 6. Schematic representation of alphaherpesvirus spread from TG neurons to the periphery during productive primary infection and upon reactivation from latency. Specific stimuli can initiate reactivation of alphaherpesviruses from latently infected TG neurons. During such reactivation events or during a productive primary infection, new virus particles are formed in the neuronal cell body (A) which are transported back to the periphery by anterograde axonal transport (either as fully assembled virus particles or as subvirion particles; see 1.3.4.) (B). Newly formed virus can exit from the axon along the axon shaft (C) and at the axon termini (D). Exit at the axon termini is associated with infection of the epithelial mucosa in which the virus replicates (E). Whether anterograde axonal transport of virus consists of the migration of fully assembled infectious virus particles or of non-infectious subvirion units, and how and where virus can spread along the axon shaft is poorly understood.

1.4.4.1. Initiation of a lytic infection differs from reactivation from latency During an acute infection, IE gene expression from the HSV genome is stimulated by incoming viral VP16 proteins and the recruitment of repressive histones to the viral DNA is

Introduction 41 ____________________________________________________________________________________________________

prevented by incoming VP22 (van Leeuwen et al, 2003; Wysocka & Herr, 2003). Furthermore, the association of the viral genome with ND10 is disrupted by the IE viral protein ICP0 which is important for transactivation of transcription, de-repression and interference with interferon-induced antiviral mechanisms (Everett & Chelbi-Alix, 2007). During latency however, the virus genome is in a largely transcriptionally repressed circular configuration associated with non-acetylated histones and in the absence of viral protein production. From this, it appears that virus reactivation from latency must overcome the repressive effects of histones and involve a mechanism of IE gene activation that can initiate in the absence of VP16. 1.4.4.1.1. Events leading to initiation of reactivation The subject of many reactivation studies is the question which consecutive events finally lead to the production of new virus particles after the latent state of infection is disrupted. Non-neuronal tissue culture cells infected with deletion mutants in VP16, ICP4, ICP0 or treated with the replication inhibitor acyclovir or derivates before infection seem to maintain the HSV genome in a very tight quiescent state as a circular molecule without any viral gene expression, including that of LATs (Scheck et al, 1986; Harris & Preston, 1991; Jamieson et al, 1995; Preston & Nicholl, 1997; Preston et al, 1997; Samaniego et al, 1997 and 1998). The only succesful way found to reactivate these quiescent genomes is the provision of ICP0 to the cells, all other methods failed (reviewed in Preston, 2000). When the quiescent infection in tissue culture cells is regarded as a model for in vivo neuronal latency, one should conclude that reactivation can only be initiated by the expression of ICP0 from the viral genome or a cellular factor must be present that mimics the action of ICP0. Studies using in vitro cultures of primary neurons or continuous neuronal cell lines, ex vivo explant models of latently infected ganglia or in vivo studies with mice to study reactivation have resulted in a somewhat different view on latency than that obtained with studies using non-neuronal cells. In neurons, the virus genome is untranscribed, except for LATs, during the latent stage of infection, but it seems to be still responsive to appropriate cellular signal transduction pathways (Preston, 2000). Whereas in tissue cultures only the provision of ICP0 can trigger the reactivation of quiescent genomes, several different stimuli have been shown to initiate the reactivation process in neurons. Different stress stimuli causing reactivation in animal models were mentioned before. Cellular changes in neurons like upregulation of cyclin dependent kinase 2 and 4 (Schang et al, 2002) and induction of the transcription factor

42 Chapter 1 ____________________________________________________________________________________________________

Bcl-3 (Tsavachidou et al, 2001), associated with the explant of latently infected ganglia lead to reactivation. In quiescently infected cultures of primary neurons, obtained by incubation with the replication inhibitor acyclovir, reactivation has been associated with stimulation or interference with cAMP dependent pathways by forskolin treatment (Hunsperger & Wilcox, 2003a), use of cAMP analogues (Smith et al, 1992) or expression of ICER from adenoviral vectors (Colgin et al, 2001). Increase in calcium levels by treatment with capsaicin (Hunsperger & Wilcox, 2003a), activation of PKC by phorbol myristate acetate treatment and activation of caspase-3 by NGF deprivation (Hunsperger & Wilcox, 2003b) also trigger reactivaton in this model system. These studies show that different triggers can cause changes in cellular protein levels and signal transduction pathways which are associated with reactivation in neurons. How this all exactly initiates reactivation is not yet clear and evidence supporting different ways of initiation of reactivation can be found in literature. A first hypothesis is that cellular changes result in the activation of promoters of IE viral genes, resulting in the accumulation of IE proteins that activate reactivation. The importance of IE proteins during reactivation has been shown by several studies. In primary cultures of TG of mice harboring latent HSV-1, reactivation could be started by supplying adenovirus vectors expressing ICP4 or ICP0 to the cells (Halford et al, 2001). Likewise, ICP0 expressed from an adenovirus vector could reactivate latent PRV in explant cultures of porcine TG (Smith & Cheung, 1998). A study by Halford & Schaffer (2001) also indicated that ICP0 is required to induce reactivation because HSV reactivation induced by heat stress was severely impaired in latently infected mice and primary neuronal cultures after infection with a ICP0mutant in comparison to WT-virus, even when the viral load of both viruses was the same. A recent study by Thompson & Sawtell (2006) confirmed that ICP0 was necessary to produce infectious virus during reactivation. However, these authors concluded that ICP0 does not play a role in the initiation of the reactivation process because, despite the fact that no infectious virus was produced, lytic proteins could be detected in one or more neurons in the majority of infected ganglia in the absence of ICP0. These results do not rule out that another viral IE protein can initiate reactivation. Another observation that seems to support the expression of IE proteins in the initiation of reactivation is that stimuli that cause reactivation correlate with a relocalisation of the transcription factor HCF from the cytoplasm to the nucleus. Because HCF, during lytic infections, forms a complex with Oct-1 and VP16 to initiate transcription, it is proposed that HCF may activate IE transcription (Kristie et al, 1999). The observation that ICP0 and ICP27 promoters can be activated in neurons and not in

Introduction 43 ____________________________________________________________________________________________________

other cell types of transgenic mice indicate that neuronal proteins can substitute for VP16 to initiate reactivation (Loiacono et al, 2002). Other studies point in the direction of cellular proteins that mimic the function of ICP0 as transactivator, rather than that ICP0 or other IE proteins are initially transcribed from the viral genome. Tal-Singer et al (1997) found that during reactivation after explant of latently infected mice ganglia, early viral transcripts were found before IE transcripts. These authors hypothesized that maybe cellular c-fos or c-jun, which are also induced after explantation, might upregulate early gene expression, thereby bypassing the need for IE proteins. Other studies indicated a possible role for a protein encoded from the LAT region to enhance lytic gene expression and substitute for ICP0 (Thomas et al, 1999 and 2002). A third hypothesis is that the signal leading to reactivation can start viral DNA replication without the onset of IE or E proteins and that DNA replication is necessary to fully activate the transcription of these proteins in this way leading to the production of infectious virus particles. This hypothesis is based mainly on the observations of Kosz-Vnenchak et al (1993) that in conditions that impaired viral DNA replication (TK- mutants or use of acyclovir) in the explant culture model, high level expression of IE or E gene expression could not be detected. These results were recently challenged however by two independent studies which showed both by ISH and RT-PCR that conditions that abbrogate DNA replication did not result in reduced levels of IE and E gene expression both in vivo and after explant (Pesola et al, 2005; Sawtell et al, 2006). As expected however, viral DNA replication is necessary to produce infectious virus during reactivation (Chen et al, 2004b). Most of the studies described above to study reactivation from latency and the initial events in this process are performed in primary neuronal cultures or in the explant model of latently infected ganglia. These models are preferred over using tissue culture cells because they use the natural target cells of the virus. Care must be taken however when the results are analysed because several disadvantages are inherent to these models. To induce a latent state of infection in primary cultures, either replication deficient viral mutants are used or acyclovir is added to the cells to inhibit virus replication. Probably, this will have an effect on the establishment of latency, leading to the possibility that the quiescent state of the virus does not completely mimic the true latent form as seen in vivo (Preston, 2000). Results obtained from studies of reactivation after explant of latently infected ganglia should also be looked at with caution because it has been shown that the explant event is a stressfull treatment for the neurons leading to both morphological and cellular changes which might effect or intervene

44 Chapter 1 ____________________________________________________________________________________________________

with the normal events leading to reactivation in vivo (Carr et al, 1998; Sawtell & Thompson, 2004). 1.4.4.1.2. Gene silencing and reactivation Besides the stimuli that can initiate reactivation via still largely unknown pathways and mechanisms, several studies describe reactivation of HSV-1 both in vivo and in vitro by stimuli that are known to interfere with the repressed state of the viral genome during latency. Sodium butyrate and trichostatin A, two known histone deacetylase inhibitors, have been shown to induce reactivation in several model systems, including in in vivo animal models, in the explant model of latently infected ganglia and in quiescently infected neuronlike PC12 cells (Arthur et al, 2001; Danaher et al, 2005; Neumann et al, 2007). Addition of these products increase acetylation of histones covering the viral genome and thereby increases the genome’s accessibility for transcription factors. Reactivation induced by neurohormones could also work via derepression of the latent genome, because it has been shown that the activated glucocorticoid receptor can bind to CREB binding protein (CBP) and in this way enhance the histone acetylation activity of CBP (Adcock et al, 2004). Another study that investigated the changes in acetylation of the viral genome during reactivation after explant of latently infected ganglia found that a decrease in LAT enhancer acetylation preceeded an increase in acetylation at the ICP0 promoter (Amelio et al, 2006). The ICP0 protein itself has also been shown to interact with class II histone deacetylases leaving open the possibility that it acts as an inhibitor of these deacetylases, in this way stimulating the conversion of the genome to an active chromatin state and so enhance further reactivation (Lomonte et al, 2004). Together these results show that the reversion of the repressive effects of histones is important during reactivation from latency. 1.4.4.2. Fate of neurons and newly produced virus upon successful reactivation It has been shown that up to 25% of the neurons in a mouse ganglion can be latently infected (Sawtell, 1997). This is in big contrast with the very low percentage of latently infected neurons that reactivate in vivo and in vitro, estimated at 1 neuron out of 1000 latently infected cells or at 1 to 6 neurons per ganglion (Shimeld et al, 1996 and 1999; Sawtell et al, 1998; Liu et al, 2001; Sawtell, 2003; Sawtell and Thompson, 2004; Danaher et al, 2006). It is not clear what is the decisive factor that makes the virus reactivate in one neuron and not in another. It

Introduction 45 ____________________________________________________________________________________________________

has however been established that there is a correlation between the viral copy number in neurons and the efficiency of reactivation (Sawtell, 1997; Sawtell, 1998, Sawtell et al, 1998). It is not known if this reflects the requirement for a threshold of genome copies necessary for reactivation or just reflects the accumulated probability that one of the genomes present in that neuron starts to reactivate. The low number of neurons in which the virus reactivates could account as a survival strategy of the virus because it is assumed that neurons do not survive a reactivation event (Shimeld et al, 1996 and 1999; Sawtell, 2003) and indicates that the immune system represses reactivation very efficiently. When a reactivation has been succesful, new virus particles are produced. Sawtell (2003) found no indications of lateral spread of new virus particles to neighbouring cells during reactivation, showing that the release of infectious virus in the ganglion in vivo is highly controlled. New virus particles are transported back to the periphery by means of axonal anterograde transport where they can cause recurrent symptoms. Also at the periphery however, virus is subject to the immune system what may limit viral spread. A first line of defence intervenes with the spread from the axon endings to the surrounding epithelial cells. Monoclonal antibodies against gB and gD have been shown to reduce transmission by 90%, probably by neutralising the virus during transmission across an intercellular gap between axon termini and epithelial cells (Mikloska et al, 1999). If the virus reaches the epithelial cells, these respond by producing type I IFNs which activate an antiviral state in these cells and attract other immune cells. There is an infiltration of CD4+- and CD8+-T cells, producing different chemokines and cytokines e.g. IFN−γ and IL-12. IFN−γ restores the MHC-I expression on infected epithelial cells and stimulates MHC-II expression on keratinocytes, thereby allowing the infected cells to be recognized by CTLs and by CD4+-T cells. Chemokines in turn attract monocytes and macrophages to the herpetic lesion (Cunningham & Merigan, 1984; Cunningham et al, 1985ab; Torseth & Merigan, 1986; Hill et al, 1995; Mikloska et al, 1996, 1999 and 2001; Koelle et al, 1998). The vigorous immune response clears the virus from the place of infection. A recent study showed that even after viral clearance, virus specific CD8+-T cells persisted in peripheral mucosa near to sensory nerve endings for more than 2 months after the herpetic lesion caused by HSV-2 reactivation was healed (Zhu et al, 2007).

46 Chapter 1 ____________________________________________________________________________________________________

1.4.5. Conclusion The results of the studies described in the previous sections clearly show that latency cannot be explained solely by the interaction beween virus and its host cell, the neuron. An important role is attributed to the host’s immune system to help control virus infection during each step of the latency/reactivation cycle. Several important aspects of the latency/reactivation cycle have been revealed during the years of intensive research, but many molecular mechanisms involved in establishment of latency, maintaining a repressed state and inducing reactivation still have to be cleared out. Differences in DNA copy number, LAT expression and responsiveness of the latent genomes have been observed which might indicate that different states of latency exist. It is hard to predict the effect of the model system used on the outcome of the experiments and the relevance of the obtained results to the in vivo situation. Besides differences that can be attributed to the model system used, the possibility that there exist different subtypes of neurons that interact differently with the virus and the immune system, leading to different outcomes of infection, also has to be considered. During recent years, several studies have drawn attention to the importance of viral genome silencing by cellular mechanisms during latent infections. Further studies will have to show the importance of this mechanism, maybe as being the main obstacle, next to the suppressive role of the immune system, that has to be overcome by the virus to reactivate from the latent state. Because in vivo studies will remain difficult to perform and to interpret, in vitro models mimicking the in vivo situation as closely as possible will be necessary to dissect different aspects of the latency/reactivation cycle at the molecular level.

Introduction 47 ____________________________________________________________________________________________________

1.5. Aims of the thesis Neurons of the trigeminal ganglion are the most important target cells for several alphaherpesviruses including herpes simplex virus 1, bovine herpesvirus 1 and pseudorabies virus. They constitute major target cells for virus spread and lifelong latent infections. Reactivation from the latent state is associated with formation of new virus particles that travel along the axon back to the periphery where they cause recurrent disease symptoms after they spread from the axon to the surrounding cells. Despite the importance of neuronal spread and the latency/reactivation cycle in the pathogenesis of alphaherpesviruses, both processes are only begun to be understood and very little is known about the molecular mechanisms controlling them. A major problem in the study of these processes is the lack of good models. Interpretation of results from in vivo studies are difficult seen the complexity of the in vivo environment. Results from in vitro studies are generally more straightforward but most in vitro models used to study alphaherpesvirus interactions with neurons do not closely mimic the in vivo situation making it difficult to predict the in vivo relevance of the obtained results. The major aim of this study was to develop an in vitro model to study alphaherpesvirus interactions with neurons in a way that mimics the in vivo situation as closely as possible. This model was then used to obtain more information about the neuronal spread of alphaherpesviruses and the conditions in which a latent infection is induced. First, a homologous in vitro two-chamber model using the porcine alphaherpesvirus PRV and porcine trigeminal ganglion neurons (the most important neuronal target cells) was developed. The model allows trigeminal ganglion neurons to become infected with PRV via retrograde axonal spread, being the natural route of infection (Chapter 2). This in vitro model was then used to study the interaction between PRV and trigeminal ganglion neurons during productive infection and to identify a novel aspect of neuronal spread, activated by virus-induced signaling pathways during the first steps of infection, being virus attachment to and entry at the axon termini (Chapter 3). Because primary neuronal cultures are very cumbersome to study signaling pathways, some of the results obtained in chapter 3 were re-examined in a more straightforward cell culture system (Chapter 4). In a last part of this study, it was examined if the latency/reactivation cycle could be reconstituted in our developed in vitro model by using interferons. Attention was paid to how this could extend the knowledge about the establishment of latent infections (Chapter 5).

_________________________________________

______________________________2.

Chapter 2: A homologous in vitro model to study interactions between alphaherpesviruses and trigeminal ganglion neurons

______________________________________Veterinary Microbiology (2006), 113, 251-255 N. De Regge, H.W. Favoreel, K. Geenen, and H.J. Nauwynck

50 Chapter 2 ____________________________________________________________________________________________________

Abstract A key aspect in the life cycle of alphaherpesviruses is their neurotropic behaviour. Sensory neurons of the trigeminal ganglion (TG) are important target cells for many alphaherpesviruses (including herpes simplex virus 1, pseudorabies virus (PRV), bovine herpesvirus 1) and constitute major sites for latent infections. Cycles of latency/reactivation and associated viral movement in and out of neuronal axons are of major importance for viral spread. The aim of this study was to develop an in vitro model that simulates the in vivo infection pattern of TG neurons by alphaherpesviruses. To this end, we developed a homologous in vitro two-chamber model using PRV and porcine TG neurons. TG of six weeks old piglets were dissociated and cultured in the inner chamber of the in vitro model, which is separated from the outer chamber by a medium- and virusimpermeable silicon barrier. Outgrowth of axons from neuronal cell bodies in the inner chamber through the silicon barrier into the outer chamber could be observed after 2 to 3 weeks of cultivation. Subsequent addition of PRV to the outer chamber resulted in exclusive infection of the TG neurons by transport of virus through the axons, subsequently giving rise to productively infected TG neurons that transmitted virus to contacting neurons and nonneuronal cells in the inner chamber. Thus, we established a unique in vitro model that mimics the natural route of alphaherpesvirus infection of TG neurons that can be used to study interactions between these viruses and this pathogenically very important cell type.

A homologous in vitro two-chamber model to study interactions between α-herpesviruses and TG neurons 51 ____________________________________________________________________________________________________

Introduction Alphaherpesviruses are a subfamily of the herpesviruses, containing closely related human and animal pathogens, including human herpes simplex virus (HSV) and varicella zoster virus (VZV) as well as important animal viruses such as the porcine pseudorabies virus (PRV), bovine herpesvirus 1 (BoHV-1) and equine herpesvirus 1 (EHV-1). These viruses mostly cause mild and recurrent diseases but in rare cases, they are associated with encephalitis. Many of the symptoms observed after infection with alphaherpesviruses are associated with their neurotropic behaviour and their ability to establish a latent infection in the peripheral nervous system of their host (Preston, 2000; Enquist et al, 2002). Sensory neurons of the trigeminal ganglion are important target cells for different alphaherpesviruses, such as PRV, HSV-1 and BoHV-1, that become infected after a primary virus replication in the epithelial cells of the upper respiratory tract (Gutekunst et al, 1980; Ackermann et al, 1982; Croen et al, 1987). The virus enters axons innervating these mucosa by fusion of the viral envelope with the axolemma near the axon terminus. The fusion is followed by retrograde transport of the capsid, and possibly a part of the tegument, to the cell body by means of microtubuleassociated fast axonal transport (Tomishima et al, 2001; Smith et al, 2004; Luxton et al, 2005). After entry of the DNA in the nucleus, either a full replication cycle is initiated, leading to the formation of new virions, or a latent infection is established (Jones, 2003). Newly produced virions, during primary infection or after reactivation, egress from TG neurons via anterograde spread along the axon and virus release at the axon terminus (Smith et al, 2001; Tomishima & Enquist, 2002). Subsequent reinfection of peripheral epithelial cells may lead to recurrent symptoms (e.g. cold sores for HSV and shingles for VZV), which ultimately may lead to infection of new hosts. Although interaction between alphaherpesviruses and TG neurons is clearly of critical importance for the life cycle of many of these viruses, many aspects of these interactions are far from fully understood. Many questions remain about the relationship between virus, TG neurons and the immune system during establishment of and reactivation from latency and about the mechanism of axonal transport and egress of these viruses. A major obstacle in solving these questions is the construction of a satisfying in vitro model to study the interaction between alphaherpesviruses and TG neurons.

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Therefore, the aim of this study was to develop an in vitro model that allows the infection of TG neurons by a homologous alphaherpesvirus via the natural retrograde axonal infection route.

Materials and methods Viruses and cells A PRV strain Becker was used for all infection experiments (Card et al, 1990). Swine testicle (ST) cells were seeded at a density of 300.000 cells/ml and cultivated in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0,1 mg/ml streptomycin, 0,1 mg/ml kanamycin and 0,3 mg/ml glutamin (basic culture medium). Medium was changed twice a week. Trigeminal ganglion neuronal cultures were established as described before (Geenen et al, 2005). Briefly, trigeminal ganglia were excised from 4 to 6 week old piglets after euthanasia with natriumpentobarbital 20% (Kela). Ganglia were dissociated by enzymatic digestion with 0,2% collagenase A (Roche). The harvested cells were resuspended in culture medium (basic culture medium without glutamine and supplemented with nerve growth factor (30 ng/ml) (Sigma)) and seeded on collagen coated cover glasses. One day after seeding, cultures were washed with RPMI (Gibco) to remove non-adherent cells and from then on, culture medium was changed three times a week. Antibodies Monoclonal mouse-anti-neurofilament-68 antibodies (Sigma) were used as a neuronal marker and were visualised with secondary Texas Red-conjugated goat-anti-mouse antibodies, purchased from Molecular Probes. Polyclonal porcine fluorescein isothiocyanaat (FITC)labelled anti-PRV antibodies (Nauwynck & Pensaert, 1995b) were used to visualise viral antigens.

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Immunofluorescence staining procedures After being washed with phosphate buffered saline (PBS), the inner and outer chamber of the two-chamber model was fixed in 100% methanol for 20 min at -20°C. All antibodies were diluted in PBS, all to a dilution of 1:100. Cells were incubated with each antibody for 1h at 37°C and all washes were performed with PBS. Confocal microscopy Confocal images were acquired using a Leica TCS SP2 laser scanning spectrum confocal system (Leica Microsystems), using an argon 488-nm laser line and a Gre/Ne 543-nm laser line to excite FITC and Texas Red respectively. Images were merged using Leica confocal software.

Results Set-up of the in vitro model To develop an in vitro model that allows TG neurons to be infected by an alphaherpesvirus of the corresponding species via the natural retrograde axonal route of infection, a two-chamber system was constructed based on the Campenot model (Campenot, 1977), and consisted of a polyallomer tube that was fixed with silicon grease on a collagen coated cover glass (Fig.1).

Figure 1. Schematic representation of the in vitro two-chamber model.

The impermeability of the silicon grease barrier for medium was checked by adding different levels of medium to the inner and outer chamber and placing the system in a CO2 incubator at

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37°C. After 14 days of incubation, fluid levels were unchanged and did not equilibrate, indicating that the barrier does not allow medium exchange between both chambers. To further check whether the silicon barrier is impermeable for virus, swine testicle cells were cultured in the inner chamber and 108 PFU of PRV were added to the outer chamber. No cytopathic effect could be observed over a 14 days period, further confirming the impermeability of the silicon barrier. In a next step, a primary porcine TG neuronal culture was establised in the inner chamber of the in vitro model. After 2 to 3 weeks of cultivation, outgrowth of newly formed axons through the silicon barrier could be observed by light microscopy (data not shown) and was confirmed by immunofluorescent stainings of the two-chamber model with anti-neurofilament antibodies, a neuronal marker (Fig.2).

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Figure 2. Confocal image of neurons in the two-chamber model after staining with anti-neurofilament antibodies (red). Arrows indicate the path of an axon growing from the neuronal cell body in the inner chamber through the silicon barrier into the outer chamber. (bar=20µm)

Pseudorabies virus infection of cultured trigeminal ganglion neurons in the in vitro model At a timepoint at which clear axon growth could be observed in the outer chamber, typically after 2 to 3 weeks of cultivation, 107 PFU of the porcine alphaherpesvirus PRV were added to the outer chamber. Two hours after inoculation, non-attached virions were removed by washing the outer chamber with PBS and new medium was added. Immunofluorescent

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stainings were used to check if inoculation of PRV in the outer chamber led to the infection of neuronal cell bodies in the inner chamber. At 24 hours post inoculation, multiple neuronal cell bodies in the inner chamber showed expression of PRV antigens, whereas non-neuronal cells were not infected (Fig.3, upper row). At 48 hours post inoculation, infection spread from the neuronal cell bodies to the surrounding monolayer of non-neuronal cells in the inner chamber (Fig.3, lower row).

Figure 3. Inner chamber of a two-chamber model, infected by addition of 107 PFU of PRV to the outer chamber, at 24 and 48 hours post inoculation. (bar=20µm)

Discussion TG neurons constitute major sites for latency and reactivation of several alphaherpesviruses. Cycles of latency and reactivation and associated movement of the virus in and out of neural axons is of major importance for the viral life cycle and alphaherpesvirus related symptoms and diseases. To study the interaction between alphaherpesviruses and these neurons, we developed an in vitro model consisting of two chambers that are physically separated by a medium- and virusimpermeable silicon barrier. Porcine TG neurons are cultured in the inner chamber where they develop axonal processes that grow through the silicon barrier into the outer chamber. Inoculation of the outer chamber with the porcine alphaherpesvirus PRV results in infection of neuronal cell bodies in the inner chamber at 24 hpi, whereas non-neuronal cells in the inner chamber do not express viral antigens at this timepoint. This shows that PRV enters the inner

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chamber via neuronal axons that are present in the outer chamber and reaches the neuronal cell body by retrograde transport where a productive infection is started. This illustrates the model’s capacity to mimic the natural route of alphaherpesvirus infection. After the initial productive infection in the neuronal cell bodies, virus was found to spread to and infect the monolayer of non-neuronal cells that surrounds the TG neuronal cell bodies in the inner chamber. This results in infection of the entire inner chamber at 48 hpi. These results show that this in vitro model fulfills all the premised aims and that it has the potential to become an interesting tool in helping to unravel some intruiging questions that still remain about the interaction between alphaherpesviruses and TG neurons.

Conclusion By making use of the porcine alphaherpesvirus pseudorabies virus, in vitro cultures of porcine trigeminal ganglion neurons and a two-chamber model, a homologous in vitro model was established that mimics the natural route of alphaherpesvirus infection of trigeminal ganglion neurons. This in vitro model can be used to study interactions between pseudorabies virus and this pathogenetically very important cell type.

Acknowledgements Nick De Regge was supported by a grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

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Chapter 3: Alphaherpesvirus glycoprotein D interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites

___________________ _____ The Journal of Cell Biology (2006), 174, 267-275 N. De Regge, H.J. Nauwynck, K. Geenen, C. Krummenacher, G.H. Cohen, R.J. Eisenberg, T.C. Mettenleiter, and H.W. Favoreel

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Abstract Alphaherpesviruses constitute closely related neurotropic viruses, including herpes simplex virus in man and pseudorabies virus (PRV) in pigs. Peripheral sensory neurons, such as trigeminal ganglion (TG) neurons, are predominant target cells for virus spread and lifelong latent infections. Here, we report that in vitro infection of swine TG neurons with the homologous swine alphaherpesvirus PRV results in the appearance of numerous synaptophysin-positive synaptic boutons (varicosities) along the axons. Non-neuronal cells that were juxtaposed to these varicosities became preferentially infected with PRV, suggesting that varicosities serve as axonal exit sites for the virus. Viral envelope protein gD was found to be both necessary and sufficient for the induction of varicosities. Inhibition of Cdc42 Rho GTPase and p38 MAP kinase signaling pathways strongly suppressed gD-induced varicosity formation. These data represent a novel aspect of the cell biology of alphaherpesvirus infections of sensory neurons, demonstrating that virus attachment/entry is associated with neuronal changes that may prepare efficient egress of progeny virus.

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Introduction Alphaherpesviruses are a subfamily of the herpesviruses, containing closely related human and animal pathogens, including human herpes simplex virus (HSV, cold sores, corneal blindness, encephalitis) and important animal viruses such as the porcine pseudorabies virus (PRV) and bovine herpesvirus 1 (BoHV-1)(respiratory symptoms, abortions and/or neurological symptoms). Many of the disease symptoms observed after infection with alphaherpesviruses are associated with their neurotropic behaviour, including their ability to establish lifelong cycles of latency and reactivation in the peripheral nervous system of their host (Preston, 2000; Enquist et al, 2002). Primary replication of most alphaherpesviruses occurs in epithelial cells of the upper respiratory tract. Sensory neurons of the trigeminal ganglion (TG) that innervate these epithelial cells are predominant target cells for HSV-1, PRV, and BoHV-1 (Gutekunst et al, 1980; Ackermann et al, 1982; Croen et al, 1987). Entrance of HSV and PRV in the axons of these sensory neurons is thought to be initiated by an interaction of the viral envelope protein gD with the receptor nectin-1, followed by fusion of the viral envelope with the axolemma, which is mediated by viral proteins gB, gD, gH, and gL (Haarr et al, 2001; Mata et al, 2001; Milne et al, 2001; Mettenleiter, 2002a; Richart et al, 2003; Spear & Longnecker, 2003). Fusion of the viral envelope with the axolemma is followed by retrograde transport of the capsid, and a part of the associated tegument, to the cell nucleus by means of microtubule-associated fast axonal transport (Tomishima et al, 2001; Smith et al, 2004; Luxton et al, 2005). After entry of the DNA into the nucleus, either a full replication cycle is initiated, leading to the formation of new virions, or a latent infection is established (Jones, 2003). Newly produced virions, during primary infection or after reactivation, are transported in the anterograde direction along the axon, followed by virus release at the axon terminus (Smith et al, 2001; Tomishima & Enquist, 2001, 2002). Recent data indicate that virus egress in axons may not be limited to the axon terminus, but also seems to occur at scattered sites along the axon shaft in a manner that remains not fully understood (Tomishima & Enquist, 2002; Ch’ng & Enquist, 2005). Despite the obvious importance of TG neurons as predominant target cells and sites of latency/reactivation events for many alphaherpesviruses, a detailed study of the interactions between alphaherpesviruses and this pathogenetically important cell type has been hampered by the lack of a functional, homologous in vitro system.

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Recently, we have established such a homologous in vitro two-chamber system, based on the ‘Campenot’ system, to study the interaction between porcine TG neurons and the porcine alphaherpesvirus PRV (Campenot, 1977; De Regge et al, 2006a). Using this in vitro model, we report here that PRV induces, via its gD envelope protein, the formation of pre-synaptic boutons (varicosities) along the axon shaft of infected TG neurons. Varicosities are swellings along neuronal axons where synaptic vesicles, mitochondria, and ER accumulate (Pannese, 1994). They are able to form synaptic contacts with contacting non-neuronal cells and other axons (Pannese, 1994), but they also seem to play an important role in non-synaptic communication in the nervous system by the release of neurotransmitters directly in the extrasynaptic space (Zhu et al, 1986; Vizi et al, 2004). We observed that non-neuronal cells aligning the axon shaft of infected TG neurons were frequently infected and the first infected non-neuronal cells were almost invariably located in close proximity to the varicosities. This suggests that virus-induced varicosities may serve as axon exit sites for the virus to infect neighbouring cells.

Materials and methods Viruses and cells Wild type PRV strain Becker, wild type PRV strain Kaplan and isogenic deletion mutants gBnull and gDnull were used (Kaplan & Vatter, 1959; Card et al, 1990; Rauh et al, 1991; Rauh & Mettenleiter, 1991). Stocks of phenotypically and genotypically complemented gBnull and gDnull viruses were grown on gB- and gD-complementing cell lines. Stocks of phenotypically and genotypically gBnull and gDnull viruses were produced by a single round of infection of phenotypically complemented gBnull and gDnull viruses on noncomplementing swine testicle (ST) cells and harvesting the progeny virus from the supernatant. Antibodies, proteins, and inhibitors Monoclonal mouse-anti-gB (1C11) and mouse-anti-gD (13D12) antibodies and polyclonal porcine FITC-labeled anti-PRV antibodies were produced as previously described (Nauwynck & Pensaert, 1995b). The monoclonal neuronal markers mouse-anti-neurofilament-68, rabbit-

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anti-neurofilament-200 and the monoclonal synapse marker mouse-anti-synaptophysin were purchased from Sigma. FITC- and Texas Red-labeled goat-anti-mouse antibodies and Texas Red-labeled goat-anti-rabbit antibodies were obtained from Molecular Probes (Invitrogen, Groningen, The Netherlands). Biotinylated sheep-anti-mouse IgG and a streptavidinbiotinylated horseradish peroxidase complex were purchased from Amersham Biosciences (Buckinghamshire, UK). Inhibitors Clostridium difficile toxin B, Y27632, Rac1 inhibitor, U0126, SB203580, and JNK inhibitor II were obtained from Calbiochem (VWR International, Leuven, Belgium). Secramine A was used as a specific inhibitor for Cdc42 as described before (Pelish et al, 2006). A truncated, soluble form of PRV-gD (PRV-gDt) (Connolly et al, 2001) and two monoclonal mouse-anti-human antibodies directed against different epitopes on the ectodomain of nectin-1 (CK6 and CK41) (Krummenacher et al, 2000) were used. Quantification of gBnull and gDnull PRV stocks The number of virus particles in the gBnull and gDnull stock was estimated by optical densitometry as described previously (Qie et al, 1999; Cheshenco and Herold, 2002). Equal volumes of a serial dilution of a wild type PRV stock with a known titer and of stocks of the genotypically and phenotypically gBnull and gDnull PRV strains were subjected to SDSPAGE under non-reducing conditions and Western blotting, followed by detection of gB or gD using monoclonal gB- and gD-specific antibodies, biotinylated secondary sheep-antimouse antibodies, streptavidin-biotinylated horseradish peroxydase complex, and detection using 3,3’-diaminobenzidine (Sigma) for gB or enhanced chemiluminiscence (ECL Western Blotting analysis system, Amersham Biosciences) for gD. All antibodies were diluted in phosphate buffered saline (PBS) with 0.1% Tween-20 (Sigma), and blots were washed three times in PBS with 0.1% Tween-20 in between different antibody incubations and after the final antibody incubation. Relative amounts of gB and gD in the gDnull and gBnull stock respectively, were compared to the amount of gB and gD present in the WT-stocks using Quantity One® 1-D Analysis Software (Biorad). Immunofluorescence staining procedures After being washed in PBS, neuronal cultures in the inner and outer chamber of the twochamber model were fixed in 100% methanol for 20 min at -20°C, except for cultures that

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were labeled for synaptophysin, which were fixed using 4% paraformaldehyde in PBS for 10 min and subsequently permeabilized in 0.2% TritonX-100 (Sigma) in PBS for 2 min. All antibodies were diluted in PBS, all to a dilution of 1:100. Cells were incubated with each antibody for 1h at 37°C and were washed two times in PBS in between all incubations steps and after the last incubation step. When necessary, nuclei were stained using Hoechst 33342 (10 µg/ml)(Molecular Probes) for 10min before the final washing steps. Cultivation, inoculation, and analysis of primary trigeminal ganglion neuronal cultures in a two-chamber model Porcine trigeminal ganglion (TG) neurons were obtained as described before (Geenen et al, 2005) and seeded in an in vitro model, based on the ‘Campenot’ system (Campenot, 1977), that allows to simulate the in vivo route of neuronal infection (De Regge et al, 2006a). In brief, porcine trigeminal ganglia were excised from 4 to 6 week old piglets and dissociated by enzymatic digestion with 0.2% collagenase A (Roche, Mannheim, Germany). The harvested cells were resuspended in culture medium (basic culture medium without glutamine and supplemented with nerve growth factor (30 ng/ml) (Sigma Chemical Compagny, St.Louis, MO, USA)) and seeded in the inner chamber of an in vitro two-chamber model. The twochamber system consists of a polyallomer tube that is fixed with silicon grease on a collagen coated cover glass inserted in a 6-well plate (Nalge Nunc International, Rochester, NY, USA). The inside of the tube forms the inner chamber, the outside forms the outer chamber. The silicon barrier prevents diffusion of medium or virus from one chamber to the other (De Regge et al, 2006a). One day after seeding, cultures were washed with RPMI (Gibco BRL, Life Technologies Inc., Gaithersburg, MD) to remove non-adherent cells and from then on, culture medium was changed three times a week. After two to three weeks of cultivation, when clear axon growth could be observed in the outer chamber, the outer chamber was inoculated with 2×106 PFU of WT-PRV or with an equivalent number of UV-inactivated-, gBnull- or gDnull-PRV particles. In some experiments, the outer chamber was incubated with soluble PRV-gD (ranging from 0.001-10 µg/ml), with antibodies directed against nectin-1 (CK6, ranging from 0.1-100 µg/ml or CK41, 100 µg/ml) or with an isotype matched (IgG1) control antibody directed to the viral envelope glycoprotein D (13D12) (100 µg/ml) (Nauwynck & Pensaert, 1995b; Krummenacher et al, 2000).

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The percentage of axons showing varicosities after different treatments was determined by fluorescent labeling using anti-neurofilament antibodies and examination of 30 axons with outgrowth in the outer chamber of different two-chamber models for the presence of multiple (>5/250 µm axon) swellings that were at least 1.5 times the diameter of the axon. Data shown represent means ± SEM of triplicate assays. FM1-43 labeling procedure The firing capacity of the induced varicosities was determined by loading the neurons with FM1-43 (Molecular Probes), basically as described before (Mizoguchi et al, 2002). After 2 to 3 weeks in culture, the outer chamber was treated for 24h with 2×106 PFU of WT-PRV, an equivalent number of UV-inactivated PRV particles or 10 µg/ml soluble PRV-gD. Then the inner chamber was washed with HBSS supplemented with 100mM KCl and 1.5mM CaCl2 for 1min. The neurons were incubated with culture medium containing 100mM KCl and 20µM FM1-43 for 10min. After being washed with HBSS for 15min, cultures were mounted on coverslips without fixation and examined by confocal microscopy. Inhibitor studies To examine the effect of different inhibitors on varicosity formation, both the inner and outer chamber of the two-chamber system were pretreated with culture medium supplemented with the respective inhibitor for 2h. Afterwards, the outer chamber was incubated with a number of UV-inactivated PRV particles equivalent to 2×106 PFU of WT-PRV, in the presence of the inhibitor. After an incubation period of 16h, the two-chamber system was methanol-fixed and stained and the percentage of axons showing varicosities was determined as described above. Quantification of single infected cells juxtaposed to varicosities Single infected cells were scored as juxtaposed to a varicosity when the signal of the viral antigens (FITC signal) contacted the varicosity (Texas Red signal), as seen in two chamber models fixed at 24hpi with 2×106 PFU of wild type PRV and stained with polyclonal FITClabeled anti-PRV antibodies and the neuronal cell marker anti-neurofilament-68 (Texas Red). 50 single infected cells were analysed and data shown represent the mean ± SEM of 4 assays.

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Confocal microscopy Stainings were analysed on a Leica TCS SP2 laser scanning spectrum confocal system (Leica Microsystems GmbH, Heidelberg, Germany) linked to a Leica DM IRBE microscope (Leica Microsystems GmbH). Images were taken using a 63x oil objective (NA 1.40-0.60) at room temperature and using Leica confocal acquisition software (Leica Microsystems GmbH). Adjustments of brightness and contrast were applied to the entire images and were done using Adobe Photoshop (Adobe Systems Inc., San Jose, CA). Statistics The mean percentages of axons displaying varicosities after the different treatments were compared with an ANOVA analysis and a LSD (least significant difference) posthoc test for a multiple comparison of means (α=0.05).

Results Induction of varicosities along the axons of PRV-infected porcine TG neurons The two-chamber system to study interactions of PRV with porcine TG neurons is mounted on a coverslip and consists of an inner chamber, in which the neuronal culture (composed of neuronal and non-neuronal cells) is seeded, and an outer chamber that are seperated from each other by a virus- and medium-impermeable silicon barrier (De Regge et al, 2006a). After two to three weeks of cultivation of trigeminal neurons in the inner chamber, axonal outgrowth through the silicon barrier into the outer chamber was detected by light microscopy. Addition of 2×106 PFU of PRV to the outer chamber resulted in exclusive infection of trigeminal neuronal cell bodies in the inner chamber as described before (De Regge et al, 2006a). Surprisingly, axons of PRV-infected neurons (24hpi) showed a massive amount of boutonlike axonal swellings, which were rarely detected on axons of non-infected cell bodies (Figure 1A). Double immunofluorescent stainings using a neuronal cell marker (neurofilament) and a marker for synaptic vesicles (synaptophysin)(Figure 1B) confirmed that the swellings are presynaptic boutons, also called varicosities.

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Labeling of the PRV-induced varicosities with FM1-43, a fluorescent marker for firing neurons, indicated that the synaptic transmission at the varicosities is intact (Figure 1C). Varicosities were formed between 3 and 6 hpi and were observed in > 70% of the axons both at early (6hpi) and late (24hpi) stages of infection. In mock-treated cultures, only 12% of the axons showed varicosities (Figure 1D).

Figure 1. Induction of varicosities along the axons of PRV-infected porcine TG neurons. (A) Confocal images of TG neurons in the inner chamber of mock-infected or PRV-infected two-chamber models at 24hpi, and stained for the neuronal marker neurofilament-68 (red) and PRV antigens (green). Scale bar = 20µm, arrows point to varicosities. (B and C) Confocal images of varicosities in the inner chamber of a two-chamber model at 24hpi with 2x106 PFU of wild type PRV and double-stained for neurofilament (red) and the synaptic vesicle marker synaptophysin (green)(B) or labeled with FM1-43 (C). Scale bar = 5µm. (D) Percentage of axons with varicosities in mock-treated or PRV-inoculated two-chamber models (3, 6 and 24hpi). Data shown represent means ± SEM of triplicate assays, percentages indicated by the same letter do not significantly differ (α=0.05).

A PRV strain that lacks the gD envelope protein is unable to induce the formation of varicosities Varicosities could be induced by UV-inactivated PRV (Figure 2B), indicating that the trigger for varicosity formation occurs early in infection, either during virus attachment or entry,

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before the onset of viral protein production. To assess whether induction of varicosities involves one of the for virus entry essential viral envelope proteins, isogenic stocks of phenotypically and genotypically gBnull and gDnull PRV were prepared. Virus stocks were obtained by inoculating phenotypically-complemented gBnull- and gDnull-PRV stocks on non-complementing ST cells, and harvesting the progeny virus from the culture supernatant. SDS-PAGE and Western Blotting confirmed that gB and gD were absent from the gBnull and gDnull PRV stocks, respectively (Figure 2A). Relative virus particle numbers in the gBnull and gDnull PRV stocks were determined as described before (Qie et al, 1999; Cheshenco and Herold, 2002), by comparing the amount of gB and gD in the deleted stocks and in serial dilutions of WT-PRV stock by optical densitometry after Western Blotting as described in Materials and Methods. Virus quantities of gBnull and gDnull PRV corresponding to the amount of particles present in 2×106 PFU of wild type PRV were added to the outer chamber of the two-chamber system and the percentages of axons displaying varicosities were determined at 24hpi. Varicosity induction by the gBnull virus was comparable to that of wild type PRV (68% vs 72%, respectively) (Figure 2B). However, when axons were inoculated with the gDnull virus, the percentage of axons with varicosities was comparable to that of the mock-treated control (14% vs 12%, respectively). As expected, neither null virus induced infection of the TG neurons, since neither was able to enter the cells (unpublished data). To exclude the possibility that the lack of synapse induction by the gDnull virus was due to an insufficient amount of virus added, the experiment was repeated with 10 fold more gDnull virus and similar results were obtained (unpublished data). In conclusion, a gDnull PRV strain is unable to induce the formation of varicosites, suggesting that virion gD triggers this process.

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Figure 2. A PRV strain that lacks the gD envelope protein is unable to induce the formation of varicosities. (A) Western blots of equal volumes of a serial dilution of a wild type PRV stock with known titer (PFU/ml) and gBnull and gDnull PRV stocks, developed with anti-gB antibodies (blot A) or anti-gD antibodies (blot B). (B) Percentage of axons with varicosities at 24h post inoculation of two-chamber models with an amount of UV inactivated wild type PRV, gBnull PRV, gDnull PRV, or wild type PRV particles equivalent to 2x106 PFU of wild type PRV. Data shown represent means ± SEM of triplicate assays, percentages indicated by the same letter do not significantly differ (α=0.05).

Recombinant PRV gD protein and nectin-1-specific antibodies induce formation of varicosities To address whether gD alone could induce varicosities, various concentrations of a truncated, soluble form of PRV-gD (PRV-gDt) (Connolly et al, 2001) were added to the outer chamber of the in vitro model and incubated with the axons for 24 hours, followed by a neurofilament staining and quantification of the number of axons that showed varicosities. Incubation resulted in a dose-dependent increase in the number of axons carrying varicosities (Figure 3A). Addition of 5 µg PRV-gDt/ml or more resulted in 60 – 70% axons with varicosities (Figure 3A), which is comparable to the percentages observed by addition of wild type PRV. Entry of alphaherpesviruses like HSV-1 or PRV into sensory neurons is believed to be mediated by interaction of viral envelope protein gD with nectin-1 (Haarr et al, 2001, Mata et al, 2001, Milne et al, 2001, Richart et al, 2003). Therefore, we hypothesized that gD-mediated induction of varicosities results from an interaction with nectin-1. To test this hypothesis, we determined whether addition of various concentrations of an antibody that binds the ectodomain of nectin-1 (CK6) (Krummenacher et al, 2000), was also able to trigger varicosity formation. As with soluble gD, a 24 hour incubation period with anti-nectin-1 Mab in the outer chamber again resulted in a clear dose-dependent response. For CK6, 100 µg/ml caused

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varicosities to form on 70% of the axons (Figure 3B), again similar to the percentage observed by addition of wild type PRV. Addition of 100 µg/ml of CK41, a monoclonal antibody directed against another epitope on the ectodomain of nectin-1, also resulted in an induction of varicosity formation (unpublished data). Addition of 100 µg/ml of an isotype matched control IgG1 antibody (13D12) did not result in an increase in the percentage of axons with varicosities compared to mock treated two-chamber systems. In conclusion, addition of recombinant gD or anti-nectin-1 antibodies to the axons of TG neurons is sufficient to induce the formation of varicosities.

Figure 3. Recombinant gD protein and nectin-1-specific antibodies induce formation of varicosities in TG neurons. Percentage of axons with varicosities in mock-treated two-chamber models or at 24h after addition of recombinant soluble PRV gD (0.001-10 µg/ml) (A), a nectin-1-specific antibody (0.1-100 µg/ml CK6) or an isotype-matched control antibody (100 µg/ml 13D12) (B) to the outer chamber of two-chamber systems. Data shown represent means ± SEM of triplicate assays, percentages indicated by the same letter do not significantly differ (α=0.05).

Cdc42 Rho GTPase and p38 MAP kinase signaling pathways are involved in alphaherpesvirus-induced varicosity formation Nectin-1 has been shown to signal via Rho GTPase signaling pathways in MDCK and L cells (Fukuhara et al, 2003, 2004). In addition, Cdc42 Rho GTPase and MAP kinase signaling pathways have been suggested to be involved in varicosity formation (Hu et al, 2004a, Nakata et al, 2005, Udo et al, 2005). Therefore, to determine which signaling pathways may be involved in PRV-induced varicosity formation, the effect of inhibitors of different signaling pathways (small Rho GTPase and MAP kinase signaling pathways) were tested for their effect on PRV-induced varicosity formation. A broad range inhibitor of small Rho GTPase

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signaling (Clostridium difficile toxin B, 50ng/ml) as well as specific inhibitors of Rho (Y27632, 30µM), Rac (Rac1 inhibitor, 100µM) or Cdc42 (secramine A, 2µM) signaling were used. The role of MAP kinase signaling was examined by using inhibitors for ERK signaling (U0126, 10µM), JNK signaling (JNK inhibitor II, 20µM) and p38 signaling (SB203580, 20µM). General inhibition of small Rho GTPase signaling as well as specific inhibition of Cdc42 signaling suppressed varicosity formation to a level that was not significantly different from mock-infected cultures, whereas specific inhibition of Rho or Rac signaling had no obvious effect (Figure 4A). Inhibition of p38 MAP kinase signaling also resulted in a strong reduction of varicosity formation, in contrast to inhibition of ERK or JNK MAP kinase signaling (Figure 4B). These data suggest that PRV activates varicosity formation via Rho GTPase (in particular Cdc42) and MAP kinase (in particular p38) dependent signaling pathways.

Figure 4. Cdc42 Rho GTPase and p38 MAP kinase signaling pathways are involved in alphaherpesvirusinduced varicosity formation. Percentage of axons with varicosities in mock-treated two-chamber models or at 16h after incubation with an amount of UV-inactivated PRV particles equivalent to 2x106 PFU of WT-PRV, in the presence of inhibitors directed against (A) small Rho GTPases (C. difficile toxin B), Rho (Y27632), Rac (Rac1 inhibitor) or Cdc42 (secramine A) or (B) ERK (U0126), JNK (JNK inhibitor II) or p38 MAP kinase signaling (SB203580). Data shown represent means ± SEM of triplicate assays, percentages indicated by the same letter do not significantly differ (α=0.05).

PRV gD-induced varicosities serve as axonal exit sites for PRV The inner chamber of the TG cultures does not consist solely of TG neurons, but also contains many non-neuronal cells, which form a monolayer in the inner chamber in which the TG neurons are dispersed. When the inner chamber of TG neuronal cultures was stained for PRV

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antigens at 24h (or later) post inoculation with WT-PRV in the outer chamber, viral antigenpositive non-neuronal cells were observed aligning the axons of PRV-infected TG neurons. Interestingly, single infected non-neuronal cells were almost invariably (88%) juxtaposed to varicosities (Figure 5). Spread of PRV from varicosities to neighbouring non-neuronal cells could not be blocked by neutralizing antibodies (unpublished data), indicating that it occurs via direct cell-cell spread. These data indicate that egress of infectious virus along the axon shaft occurs specifically at varicosities.

Figure 5. Varicosities serve as axonal exit sites for PRV. (A) Confocal images of TG neuronal cultures in the inner chamber of PRV-infected two-chamber systems at 24hpi and stained for neurofilament-68 (red), PRV antigens (green) and nuclei (blue). Scale bar = 5µm, arrows point to varicosities. Graph in (B) shows the percentage of single infected cells that are juxtaposed to varicosities, calculated compared to the total number of single infected cells. Data shown represent means ± SEM of 4 assays.

Discussion The neurotropic behaviour of alphaherpesviruses is of crucial importance for the pathogenicity of these viruses, allowing them to establish lifelong latency, cause central

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nervous disorders, encephalitis and recurrent disease. Neurons of the trigeminal ganglion (TG) represent crucial target neurons for many alphaherpesviruses, including HSV-1, PRV, and BoHV-1. The exact cell biology underlying the interactions of alphaherpesviruses with neurons, especially TG neurons, remains far from fully understood. Here, we used an in vitro model to study the interaction of an alphaherpesvirus (PRV) with TG neurons of its corresponding host (the pig). We report a novel aspect of the cell biology of alphaherpesvirus interaction with TG neurons. We found that the interaction of PRV with axons of porcine TG neurons triggers the formation of synaptic boutons (varicosities) along the axons of these neurons. To our knowledge, this is the first report describing that a virus infection stimulates the formation of varicosities. In addition, we show that the viral envelope protein gD is responsible for the induction of varicosities, probably via an interaction with the entry receptor nectin-1 or nectin-like molecules, that Cdc42 Rho GTPase and p38 MAPK signaling pathways are involved, and that virus egress along the axon shaft of infected TG neurons occurs frequently and specifically at these varicosities. These observations open the intruiging possibility that the virus has evolved a strategy to facilitate spread of progeny virus from TG neurons by inducing varicosities at the time of virus attachment and entry in axons. An important question is how exactly PRV induces the formation of varicosities in porcine TG neurons. We found that addition of UV-inactivated PRV (which is able to adhere to and penetrate cells but does not start up viral gene expression) or a strain of PRV that lacks the envelope protein gB (which is able to adhere to but not to penetrate cells) to the axons of TG neurons still resulted in varicosity formation. This shows that varicosity formation is triggered during virus attachment to the axons and does not require infection. Interestingly, a PRV strain that lacks gD in its envelope (which is also able to adhere to but not to penetrate cells) did not induce varicosities, demonstrating an involvement of gD in this process. Moreover, addition of recombinant soluble gD to the axons of TG neurons was sufficient to trigger varicosity formation. Together, these data indicate that interaction of PRV envelope protein gD with axons of TG neurons during virus attachment provides the trigger necessary for subsequent formation of varicosities. How does the interaction of gD with the surface of axons lead to varicosity formation? Three classes of receptors for alphaherpesvirus gD proteins have been described to date: one belongs to the tumor necrosis factor receptor family (i.e. HVEM), another class belongs to the immunoglobulin superfamily (including nectins 1 and 2), and another type of receptor consists of modified heparan sulfate (Montgomery et al, 1996; Cocchi et al, 1998; Geraghty et

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al, 1998; Warner et al, 1998; Shukla et al, 1999; Spear & Longnecker, 2003). Nectin-1 has been suggested to serve as the gD receptor on sensory neurons (like TG neurons) for both HSV and PRV (Haarr et al, 2001; Mata et al, 2001; Milne et al, 2001; Richart et al, 2003). Nectins are cell adhesion molecules that play important roles in the formation of many types of cell-cell junctions, including synapses (Takai et al, 2003b). We found that the addition of two different nectin-1-specific antibodies to the axons of TG neurons (as a surrogate ligand for nectin-1 instead of gD) both resulted in the formation of varicosities. This suggests that the interaction between gD and nectin-1 on the axons of TG neurons provides the trigger that ultimately culminates in the formation of varicosities. This would be consistent with observations by Mizoguchi et al (2002), who showed that stimulation of nectin-1 in mouse hippocampus neurons resulted in a substantial increase in the number of synaptophysinpositive varicosities. However, at present, we cannot rule out that other nectins or nectin-like molecules, in addition to, or instead of nectin-1 might be relevant to the gD-mediated induction of varicosities. Porcine nectin-1 is thus far the only porcine entry receptor that has been reported for PRV (Milne et al, 2001), but different human forms of the nectin family, like nectin-1, nectin-2, and necl-5 (poliovirus receptor), have been reported to be gD-binding entry receptors for PRV (Geraghty et al, 1998, Connolly et al, 2001). In addition to a crucial role of the interaction between gD and nectin-1 and/or other members of the nectin family for the PRV-induced formation of varicosities, we also found that Cdc42 Rho GTPase as well as p38 MAPK signaling pathways are involved. Inhibition of these signaling pathways strongly suppressed PRV-induced varicosity formation. These data are consistent with recent indications that the Cdc42 (but not Rho or Rac) signaling pathway is involved in serotonin-induced varicosity formation in sensory neurons (Udo et al, 2005) and that MAP kinase signaling pathways such as p38 signaling pathways may play important roles during development of varicosities (Hu et al, 2004a, Nakata et al, 2005). Further in line with our current observations, it has been reported earlier that nectin-1 may signal through Rho GTPase pathways, such as Cdc42 and Rac, in MDCK and L cells (Fukuhara et al, 2003, 2004). Our current findings that an alphaherpesvirus, via its envelope glycoprotein D, stimulates formation of synaptophysin-positive varicosities and that this process involves Cdc42 and p38 MAPK signaling may therefore constitute a valuable tool to further dissect the underlying molecular mechanisms of varicosity formation and the role of nectin-1 and other nectin-like molecules herein. Alphaherpesviruses have evolved different strategies to modulate the host cell in order to facilitate virus spread. These mechanisms include gE-mediated targeting of virus particles to

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cell-junctions for spread in polarized cells (Collins & Johnson, 2003; Wisner & Johnson, 2004) and US3-induced formation of virus-containing cell projections that are associated with enhanced intercellular spread (Favoreel et al, 2005). Although future in vivo experiments will be required to fully delineate the consequences of our current findings for alphaherpesvirus pathogenesis, we propose that induction of axonal varicosities by PRV early in infection of TG neurons may be part of the viral strategy to later promote efficient spread. Varicosity-mediated spread may lead to egress of alphaherpesviruses along the axon shaft, but may also enhance spread of the virus to mucosal surfaces, a crucial step in alphaherpesvirus pathogenesis. Trigeminal sensory nerve fibers may extend beyond the basement membrane to nearly reach the epithelial surface (Finger et al, 1990). These intra-epithelial terminal regions of the axon have penetrated the basement membrane, lost their myelin sheath and are prone to varicosity formation. Strings of varicosities have been reported to occur in these intraepithelial terminal areas of the TG axons in vivo (Kondo et al, 1992, Ibuki et al, 1996). The exact function of alphaherpesvirus egress along the entire length of axon shafts is not understood, but PRV egress along axons shafts has clearly been demonstrated in vivo in rats, and was found to occur via direct cell-cell spread, which is in line with our current findings (Tomishima & Enquist, 2002). Interestingly, this axonal egress in vivo as well as axonal egress in vitro was found to occur at scattered sites (Tomishima & Enquist, 2002, Ch’ng & Enquist, 2005). Given our current finding that axonal egress almost exclusively occurs at varicosities, we hypothesize that these scattered sites may correspond to varicosities. In further support of this, during the review process of the current manuscript, a report appeared by Miranda-Saksena et al which suggests that axonal egress of HSV-1 occurs via varicosities in human fetal dorsal root ganglia neurons in vitro (Miranda-Saksena et al, 2006). Transport of progeny herpesvirus particles in the axon is believed to either occur via secretory vesicles containing fully matured virions or as subvirion particles, where capsids and envelope proteins are transported separately and only assemble into mature virions at synapses (Tomishima & Enquist, 2002). To our opinion, both scenarios can lead to specific axonal egress at varicosities, since these structures have been shown to accumulate secretory vesicles and may also constitute functional synapses (Santos et al, 2001). Virus-induced varicosities endured for at least several days (>72h)(unpublished data). It remains to be determined whether and when they disappear after the initial infection. Although speculative at this time, it is possible that the virus is able to re-induce varicosities over and over again when needed during latency/reactivation cycles. Reactivations can lead to

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production and release of infectious virus, which in turn may attach to and enter new TG neurons, thereby inducing varicosity formation and further promoting virus spread. It is possible that virus-induced formation of varicosities has consequences beyond virus spread. At least a subpopulation of synaptophysin positive varicosities function as sites for neurotransmitter release along the axon of different types of neurons (Kohara et al, 2001; Pennuto et al, 2002; Morgenthaler et al, 2003). In line with this, we found that the virusinduced varicosities stain positive for FM1-43, characteristic for intact synaptic transmission. Synaptic transmission was also intact in varicosities induced by UV-inactivated PRV or recombinant gD of PRV (unpublished data). In this context, it is interesting to note that alphaherpesvirus infections have been associated with hyper-excitability of neurons, possibly involved in acquired epilepsy following HSV encephalitis (Chen et al, 2004a). Future investigations will be designed to further unravel whether the PRV-induced formation of synaptically effective varicosities may lead to changes in excitability of neurons. In conclusion, we have found that entry of an alphaherpesvirus in neurons of the trigeminal ganglion of its natural host is associated with the induction of synaptic varicosities along the axons. Virus-induced varicosity induction depends on viral envelope protein gD and on Cdc42 Rho GTPase and p38 MAP kinase signaling pathways and the virus uses the varicosities as axonal egress sites to spread to neighbouring cells.

Acknowledgments The authors would like to thank Carine Boonen, Lieve Sys, Chantal Vanmaerke and Nele Dennequin for excellent technical assistance and Kevin Braeckmans for help with confocal microscopy. We also would like to thank the Kirchhausen lab (Harvard Medical School) and the Hammond lab (University of Louisville) for the kind gift of secramine A that was synthesized by B. Xu and G.B. Hammond of the University of Louisville. This research was supported by a Concerted Research Action of the Research Council of Ghent University and research grant G.0227.04 from the Research Foundation-Flanders (FWO-Vlaanderen). N.D.R. is the recipient of a grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). C.K. is supported by an award from the University of Pennsylvania Research Foundation. This investigation was supported in part by Public Health Service grants NS-36731 from the National Institute of Neurological Disease

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and Stroke (R.J.E. and G.H.C.), AI-056045 and AI-18289 from the National Institute of Allergy and Infectious Diseases (to R.J.E. and G.H.C. respectively).

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Chapter 4: Transfection of pseudorabies virus glycoprotein D in nectin-1 expressing CHO cells results in Rho GTPase-dependent formation of filopodia-like structures

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______Manuscript in preparation

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Abstract The interaction of glycoprotein D (gD) with one of its cellular entry receptors is crucial for succesful entry of nearly all alphaherpesviruses. Nectin-1, a cell-cell adhesion molecule, is known to be an important entry receptor for several alphaherpesviruses, including HSV-1 and PRV. In a previous report, we described that the interaction between pseudorabies virus (PRV)-gD and trigeminal ganglion (TG) neurons during virus entry activated intracellular signaling dependent on small Rho GTPases and MAP kinases resulting in the formation of varicosities, probably upon interaction of gD with nectin-1. This suggests that nectin-1 transduces signaling upon interaction with gD. Besides an interaction during entry, gD and nectin-1 have also been shown to interact during a later step in infection, when newly synthesized HSV-gD on the surface of the infected cell can interact with nectin-1 present on contacting cells (Krummenacher et al, 2003). In the current study, we evaluate whether transfection of gD in nectin-1-expressing CHO cells may serve as a model for gD-nectin-1 induced varicosities in order to facilitate the unraveling of the underlying signaling pathways. Surprisingly, we find that the gD – nectin-1 interaction in the CHO model induces a different kind of signaling, transduced by the gD protein and not by nectin-1. We report that transfection of endocytosis deficient forms of PRV-gD in CHO cells expressing nectin-1 induces the formation of filopodia-like structures in about 38% of the gD-expressing transfected cells, while this is only observed in about 10% of PRV-gD transfected CHO cells lacking nectin-1. Wild type PRV-gD also induced filopodia-like structures but to a lesser extent (21%). Co-seeding experiments showed that the induction of filopodia-like structures is dependent on an interaction between gD and nectin-1 in trans and, surprizingly, that the trigger appears to be passed on to the cell via gD and not via nectin-1. By using specific inhibitors of small Rho GTPases and MAP kinases and performing cotransfections with PRV-gD and dominant negative forms of small Rho GTPases, it was shown that signaling via Rac1 and Cdc42 small Rho GTPases and ERK-MAP kinase is critical in the formation of gD – nectin-1 induced filopodia-like structures. In conclusion, we found that transfection of PRV-gD in nectin-1 expressing CHO cells results in the formation of filopodia-like structures dependent on a trans interaction between newly produced PRV-gD and nectin-1. Rac1 and Cdc42 small Rho GTPase and ERK-MAP kinase signaling appears to be involved in this process. Although the signaling molecules involved are largely similar, the gD – nectin-1 induced signaling is very different in CHO cells versus

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neurons. The signaling cascade induced by the gD – nectin-1 interaction in transfected CHO cells appears to be initiated by gD and not by nectin-1. Therefore, transfection of gD in nectin-1 expressing CHO cells is not a good model to study signaling pathways underlying gD – nectin-1 mediated varicosity formation in TG neurons. However, it may be interesting to evaluate further whether this new aspect of gD – nectin-1 signaling in CHO cells may have biological significance.

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Introduction Viruses have evolved different, often fascinating, interactions with host cells to create an optimal niche for virus replication, spread, and persistence. A rather poorly understood but potentially interesting type of virus-cell interactions are viral interactions with cellular signal transduction pathways. Cellular signal transduction cascades are involved in a plethora of cellular activities, including but certainly not limited to cellular responses to extracellular stimuli (e.g. stress responses, cytokine responses, growth factor responses), cytoskeletal rearrangements, cell division, apoptotic cell death, and gene regulation. Because of their involvement in a vast majority of cellular processes, it is likely that viruses have evolved different interactions with these signaling networks. Alphaherpesviruses are large DNA viruses with a lipid envelope and represent the largest subfamily of the herpesviruses. They contain closely related viruses of man and animal, including herpes simplex virus (HSV) and varicella-zoster virus in man, pseudorabies virus (PRV) in swine, and bovine herpesvirus 1 (BoHV1) in cattle. Viral proteins in the lipid outer layer of herpesvirus particles, the envelope proteins, are of criticial importance during entry in host cells. Entry of nearly all alphaherpesviruses in host cells critically relies on envelope proteins gB, gD, gH, and gL (Reske et al, 2007). Recently, we have found that during entry of PRV in swine trigeminal ganglion neurons, important target cells for many alphaherpesviruses, the interaction between the gD envelope protein and its receptor on these sensory neurons triggers intracellular signaling pathways depending on small Rho GTPases and MAP kinases that culminate in the formation of synaptic varicosities that may serve as virus egress sites of progeny virus particles (De Regge et al, 2006b). Several cellular receptors of the gD protein have been reported (Spear, 2004). The cellular adhesion molecule nectin-1 has been put forward as the principal gD receptor in neurons (Haarr et al, 2001; Mata et al, 2001; Milne et al, 2001; Richart et al, 2003; Simpson et al, 2005; Ono et al, 2006). Nectin-1 is a Ca2+-independent Ig-like cell-cell adhesion molecule that is found at adherens junctions and that belongs to the nectin family. Just like the other three members of the nectin family, nectin-1 has three Ig-like loops in its extracellular region, one transmembrane segment and one cytoplasmic region. Each nectin forms first homo-cisdimers and then homo- or hetero-trans-dimers through the extracellular region in a Ca2+independent manner causing cell-cell adhesion (Takai & Nakanishi, 2003; Takai et al, 2003a). Importantly, trans interactions between nectins have been reported to be able to activate Rho

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GTPase signaling pathways, leading to filopodia in fibroblasts and epithelial cells and varicosities in neurons (Kawakatsu et al, 2002; Mizoguchi et al, 2002; Takai et al, 2003b). Nectin-1 is also involved in the activation of ERK-MAP kinase signaling that leads to the production of loricrin in keratinocytes (Wakamatsu et al, 2007). We found that activation of nectin-1 on TG neurons via nectin-1 specific antibodies resulted in the formation of varicosities, similar as what we observed after addition of PRV or recombinant gD (De Regge et al, 2006b). Together, this suggests that the interaction between gD and nectin-1 results in Rho and MAP kinase-dependent intracellular changes. In support of this, a recent report has indicated that addition of HSV to host cells may result in Rho GTPase signaling, leading to the formation of filopodia. This process was found to depend on nectin-1, since it occurred in nectin-1-expressing CHO cells, but not in normal CHO cells that lack detectable nectin-1 expression (Clement et al, 2006). The aim of the current study was to further clarify cellular morphological changes and intracellular signaling induced by the interaction between gD and nectin-1. To this end, we analysed the effect of transfection of PRV gD in CHO cells that either express or lack nectin1. Surprisingly, we find that the gD – nectin-1 interaction in the CHO model induces a very different type of signaling than the gD – nectin-1 interaction in neurons, although similar signaling molecules appear to be involved.

Materials and Methods Cells Wild type CHO cells (wt-CHO) and nectin-1 expressing CHO cells (CHO-nectin-1) were provided by P.G. Spear (Northwestern University, Chicago, USA). All CHO cell lines were grown in F12 medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0,1 mg/ml streptomycin, 0,1 mg/ml kanamycin, 0.3 g/l glutamine and 1mM sodium pyruvate.

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Vectors expressing wild type or mutant PRV-gD and dominant negative forms of Rac1 and Cdc42 Mammalian expression vectors pBudCE4.1 encoding i) wt-PRV-gD, ii) PRV-gD containing a point mutation (YRLA) in the endocytosis motif in the cytoplasmic tail (endo--gD) and iii) PRV-gD in which a premature translation termination codon was inserted resulting in a truncation of almost the entire cytoplasmic domain, except for the first 5 amino acids (gDtrunc) were used and were described before (Ficinska et al, 2005). dLZRS-IRES-EGFP vectors (which are a modified form of the LZRS-IRES-EGFP vector (Heemskerk et al, 1997)) expressing dominant negative forms of human Rac1 and Cdc42 fused to an E-tag marker (Sander et al, 1999; Schotte et al, 2004) were a kind gift of C. Stove (Ghent University, Belgium). Antibodies, inhibitors and chemicals Monoclonal mouse-anti-gD (13D12) antibodies were produced at our laboratory as previously described (Nauwynck & Pensaert, 1995b). Monoclonal swine-anti-gD antibodies were a gift from Dr. S. Brockmeier (Midwest Area National Animal Disease Center, Ames, Iowa) and monoclonal mouse-anti-human nectin-1 antibodies (CK6; Krummenacher et al, 2000) were a gift of Dr. C. Krummenacher. Monoclonal mouse-anti-E-tag was purchased from Amersham Biosciences (Buckinghamshire, UK), monoclonal mouse-anti-β-actin was purchased from Sigma and polyclonal rabbit-anti-tubulin was purchased from Abcam (Camebridge, UK). Phalloidine-Texas Red, FITC- and Texas Red-labeled goat-anti-mouse antibodies and Texas Red-labeled goat-anti-rabbit antibodies were obtained from Molecular Probes (Invitrogen, Groningen, The Netherlands) and FITC-labeled goat-anti-swine antibodies were obtained from Jackson I.R. laboratories (West Growe, PA, USA). CellTracker Green CMFDA was purchased from Molecular Probes. LipofectaminTM was obtained from Invitrogen. Inhibitors Clostridium difficile toxin B, Y27632, Rac1 inhibitor, U0126, SB203580, and JNK inhibitor II were obtained from Calbiochem (VWR International, Leuven, Belgium). Secramine A was used as a specific inhibitor for Cdc42 (Pelish et al, 2006) and was a kind gift of the Kirchhausen and the Hammond lab (Harvard Medical School and University of Louisville).

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SDS-PAGE and Western Blotting Equal amounts of lysed wt-CHO and CHO-nectin-1 cells were subjected to SDS-PAGE under reducing conditions and Western blotting, followed by detection of nectin-1 and β-actin using monoclonal nectin-1- and β-actin-specific antibodies, biotinylated secondary sheep-antimouse antibodies, streptavidin-biotinylated horseradish peroxydase complex, and detection using enhanced chemiluminiscence (ECL Western Blotting analysis system, Amersham Biosciences). All antibodies were diluted in phosphate buffered saline (PBS) with 0.1% Tween-20 (Sigma), and blots were washed three times in PBS with 0.1% Tween-20 in between different antibody incubations and after the final antibody incubation. Transfection protocol Subconfluent wt-CHO cell and CHO-nectin-1 cell monolayers were (co)transfected with vectors encoding wt-PRV-gD, mutant PRV-gD ORF’s and/or vectors encoding dominant negative forms of Rac1 and Cdc42 by use of LipofectamineTM reagent according to manufacturer’s instructions. Co-seeding experiments wt-CHO cells or CHO-nectin-1 cells were seeded at low density (25.103 cells/ml) and transfected with gDtrunc at 24 h post seeding. At 3h post transfection, either 4.105 wt-CHO cells or CHO-nectin-1 cells labeled with CellTracker were added. Labeling with CellTracker was done before the cells were added to the transfected cells by incubation of dissociated cells for 15 min in F12 medium containing 20µM CellTracker Green. Cells were fixed at 48 h post transfection. For quantification, only cells expressing PRV-gD that were not labeled with CellTracker were analysed. Inhibitor studies At 3 h post transfection of subconfluent monolayers of CHO-nectin-1 cells with endo--gD, inhibitors were added to the transfected cells at the indicated concentrations and remained with the cells till the end of the experiment. Cells were fixed at 24 h post transfection.

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Immunofluorescent staining procedures At 24 or 48h post transfection, cells were fixed with 3% paraformaldehyde for 15 min and permeabilised with 0.2% Triton-X 100 for 2 min. For tubulin stainings, both fixation and permeabilisation were performed in cytoskeleton stabilizing buffer (10mM Pipes buffer, 150mM NaCl, 5mM EGTA, 5mM MgCl2 and 5mM glucose monohydrate). All antibodies were diluted in PBS, all to a dilution of 1:100 except for anti-E-tag (1:500) and 13D12 (1:30). Cells were incubated with each antibody for 1h at 37°C and were washed two times in PBS in between all incubations steps and after the last incubation step. Confocal microscopy Stainings were analysed on a Leica TCS SP2 laser scanning spectrum confocal system (Leica Microsystems GmbH, Heidelberg, Germany) linked to a Leica DM IRBE microscope (Leica Microsystems GmbH). Images were taken using a 63x oil objective (NA 1.40-0.60) at room temperature and using Leica confocal acquisition software (Leica Microsystems GmbH). Adjustments of brightness and contrast were applied to the entire images and were done using Adobe Photoshop (Adobe Systems Inc., San Jose, CA). Quantification and statistics Quantification of the percentage of transfected cells showing filopodia-like structures in the different experiments was done by analysis of 200 gD-transfected cells, that were labeled with mouse-anti-gD monoclonal antibodies and a fluorescently marked secondary antibody, for the presence of filopodia-like structures. Stainings were analysed by fluorescence microscopy. Data shown represent means ± SEM of triplicate assays. The mean percentages of transfected cells showing filopodia-like structures after transfection with different forms of PRV-gD and after different inhibitor treatments were compared with an ANOVA analysis and a Tukey posthoc test for a multiple comparison of means (α=0.05).

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Results Transfection of PRV-gD in nectin-1 expressing CHO cells induces formation of filopodia-like structures The absence of nectin-1 in wt-CHO cells and its presence in CHO-nectin-1 cells was confirmed by SDS-PAGE and Western Blotting (Figure 1A). The PRV-gD gene was transfected into wt-CHO or CHO-nectin-1 cells and cells were analyzed for morphological changes at 24 h post transfection. Because we described earlier that the cytoplasmic tail of wtPRV-gD contains an endocytosis motif (YRLL) that drives efficient endocytosis of gD from the plasma membrane in gD-transfected cells (Ficinska et al, 2005), transfections were done with a plasmid carrying a single point mutation (YRLA) in the endocytosis motif (endo--gD) to ensure high levels of gD expression on the cell surface of transfected cells. Transfection of endo--gD in CHO-nectin-1 cells resulted in 38.2 ± 2.1% of transfected cells showing filopodia-like structures, compared to only 11.9 ± 1.8% in wt-CHO cells expressing endo--gD (stainings with Phalloidin-Texas Red to visualise actin showed that 5.3 ± 2.2% of untransfected wt-CHO cells showed the presence of filopodia-like structures (data not shown)) (Figure 1B and 2).

Figure 1. Transfection of endo--PRV-gD in nectin-1 expressing CHO cells induces formation of filopodialike structures. (A) Western blot of equal amounts of lysed wt-CHO and CHO-nectin-1 cells, developed with anti-nectin-1 and anti-β-actin antibodies. (B) Confocal image of CHO-nectin-1 cells fixed at 24 h post transfection with endo--gD and stained for gD (green). (bar = 2µm)

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Transfection of wt-gD also resulted in induction of filopodia-like structures in CHO-nectin-1 cells, albeit to a lesser extent (21.3 ± 2.7%). Transfection of a gD recombinant carrying a premature stop codon removing nearly the entire cytoplasmic domain of gD (gDtrunc), including the endocytosis motif, in CHO-nectin-1 cells also resulted in the formation of filopodia-like structures in 38.4 ± 2.8% of transfected cells. Remarkably, induction of filopodia-like structures was only observed in the transfected cells, not in cells contacting the gD expressing cells. Immunofluorescent stainings with antibodies directed to tubulin to visualize microtubules showed that no microtubules were present in the cell extensions, supporting their filopodia-like nature. In conclusion, transfection of endocytosis-deficient and to a lesser extent wild type PRV-gD induces formation of filopodia-like structures in CHO-nectin-1 cells while these are only present in a minority of transfected wt-CHO cells, indicating that the interaction between gD and nectin-1 triggers the formation of these structures.

Figure 2. Percentage wt-CHO cells and CHO-nectin-1 cells showing filopodia-like structures at 24 h post transfection with vectors expressing different PRV-gD-ORF’s. Percentage of wt-CHO cells and CHO-nectin1 cells showing filopodia-like structures at 24 h post transfection with expression vectors encoding for i) wt-gD, ii) mutant gD that lacks a functional endocytosis motif in its cytoplasmic tail (endo--gD) and iii) mutant gD lacking the cytoplasmic tail except for the 5 first amino acids (gDtrunc). Data shown represent means ± SEM of triplicate assays. Percentages indicated by the same letter do not significantly differ (α = 0.05).

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Interaction between PRV-gD and nectin-1 in trans induces formation of filopodia-like structures Co-seeding experiments were performed to examine whether the observed formation of filopodia-like structures in CHO-nectin-1 cells after transfection with gDtrunc are dependent on a cis or trans interaction between PRV-gD and nectin-1. Transfection of wt-CHO cells seeded at low density with gDtrunc, followed by addition of wt-CHO or CHO-nectin-1 cells at 3h post transfection resulted in respectively 11.5 ± 1.2% and 23.0 ± 0.8% of transfected cells showing filopodia-like structures at 48 h post transfection (Figure 3 and 4). When gDtrunc was transfected in CHO-nectin-1 cells in a similar experiment, 11.1 ± 1.5% of transfected cells showed filopodia-like structures at 48h post transfection when wt-CHO cells were added, while this was 19.8 ± 1.4% after addition of CHO-nectin-1 cells. These data suggest that the interaction between gD and nectin-1 in trans induces the formation of the filopodia-like structures in the gD transfected cells. Since the filopodia-like structures were only observed in the gD-expressing cells, this suggests that gD induces an intracellular signal upon interaction with nectin-1 on an adjacent cell. This is different from our earlier observation on gD – nectin-1 mediated varicosity formation in TG neurons (De Regge et al, 2006b), where the signal is given to the cell via nectin-1. Thus, nectin-1 expressing CHO cells are not a good model to study signaling pathways underlying gD -

nectin-1 mediated

varicosity formation.

Figure 3. Interaction between PRV-gD with nectin-1 in trans induces the formation of filopodia-like structures. Confocal image of a wt-CHO cell transfected with gDtrunc (red) and surrounded by CHO-nectin-1 cells (green). CellTracker green-labeled CHO-nectin-1 cells were added at 3h post transfection of wt-CHO cells with gDtrunc and cells were fixed at 48h post transfection. (bar = 2µm)

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Figure 4. Percentages of CHO cells transfected with gDtrunc showing filopodia-like structures after a cis and/or trans interaction with nectin-1. Sparsely seeded wt-CHO and CHO-nectin-1 cells were transfected with gDtrunc and, at 3 h post transfection, the equivalent of a monolayer of wt-CHO or CHO-nectin-1 cells was added. Percentages of gD transfected cells showing filopodia-like structures was determined at 48 h post transfection. Data shown represent means ± SEM of triplicate assays. Percentages indicated by the same letter do not significantly differ (α = 0.05).

Rac1 and Cdc42 small Rho GTPases and ERK-MAP kinase are involved in formation of filopodia-like structures upon interaction between PRV-gD and nectin-1 Despite the differences between gD – nectin-1 induced signaling in CHO cells versus TG neurons, we went further to determine the signaling molecules involved in gD – nectin-1 mediated filopodia-like structure formation in CHO cells. Rho GTPase and MAP kinase signaling have been shown to be involved in filopodia formation and in nectin-1 and gDnectin-1 mediated signaling (Hall, 1998; Kawakatsu et al, 2002; De Regge et al, 2006b). Therefore, we determined if small Rho GTPases and MAP kinases are also involved in the observed induction of filopodia-like structures after transfection of endo--gD in CHO-nectin-1 cells. First, we examined the effect of different inhibitors of small Rho GTPases on the formation of filopodia-like structures after transfection of endo--gD in CHO-nectin-1 cells (Figure 5). A broad range inhibitor of small Rho GTPase signaling (400 ng/ml Clostridium difficile toxin B) as well as specific inhibitors of Rho (30 µM Y27632), Rac1 (100 µM Rac1 inhibitor), or Cdc42 (0.8 µM secramine A) signaling were used. A significant reduction in the number of transfected CHO-nectin-1 cells showing filopodia-like structures was observed after general inhibition of small Rho GTPases (16.9 ± 5.8%) and specific inhibition of Rac1 (19.4 ±1.4%)

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and Cdc42 (17.9 ± 1.5). Specific inhibition of Rho (41.0 ± 2.0%) did not result in a reduction of CHO-nectin-1 cells showing filopodia-like structures after transfection with endo--gD when compared to transfection of endo--gD in untreated CHO-nectin-1 cells. We also tested the effect of lower concentrations of these inhibitors (100 ng/ml toxin B; 7,5 µM Y27632; 25 µM Rac1 inhibitor and 0,2 µM secramine A) on the formation of filopodia-like structures after transfection of endo--gD in CHO-nectin-1 cells. These concentrations caused similar but less pronounced effects as those observed for the higher concentrations (data not shown). The role of Rac1 and Cdc42 in the formation of filopodia-like structures after interaction between gD and nectin-1 in CHO-nectin-1 cells was confirmed by performing co-transfections with plasmids encoding endo--gD and plasmids encoding dominant negative (DN) forms of Rac1 or Cdc42. Only 14.0 ± 1.9% and 12.4 ± 3.0% of CHO-nectin-1 cells expressing both endo-gD and respectively DN-Rac1 or DN-Cdc42 had filopodia-like structures which is comparable to the percentage observed upon endo--gD transfection in wt-CHO cells.

Figure 5. Rac1 and Cdc42 small Rho GTPase signaling is involved in formation of filopodia-like structures after transfection of endo--gD in CHO-nectin-1 cells. Percentage of wt-CHO cells and CHOnectin-1 cells showing filopodia-like structures at 24 h post transfection with endo--gD in untreated cells or when inhibitors directed to small Rho GTPases (C. difficile toxinB), Rho (Y27632), Rac1 (Rac1 inhibitor) or Cdc42 (secramine A) were added at 3 h post transfection or when endo--gD was co-transfected with dominant negative forms of Rac-1 or Cdc42. Data shown represent means ± SEM of triplicate assays. Percentages indicated by the same letter do not significantly differ (α = 0.05).

The involvement of MAP kinase signaling in filopodia-like structure formation by gD – nectin-1 interaction in endo--gD transfected CHO-nectin-1 cells (Figure 6) was examined by the use specific inhibitors of ERK (10 µM U0126), JNK (20 µM JNK inhibitor II), and p38 (20 µM SB203580) signaling. Specific inhibition of ERK resulted in a significant reduction in

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the number of transfected cells having filopodia like structures (14.9 ± 2.4%), while inhibition of JNK (42.5 ± 6.3%) and p38 (45.9 ± 4.3) appeared to have no effect. In conclusion, these data suggest that Rac1 and Cdc 42 small Rho GTPases and ERK-MAPK signaling are involved in the formation of filopodia-like structures observed after transfection of endo--gD in CHO-nectin-1 cells.

Figure 6. ERK-MAP kinase signaling is involved in formation of filopodia-like structures after transfection of endo--gD in CHO-nectin-1 cells. Percentage of wt-CHO cells and CHO-nectin-1 cells showing filopodia-like structures at 24 h post transfection with endo--gD in untreated cells or when inhibitors directed to ERK (U0126), p38 (SB203580) or JNK (JNK inhibitor II) MAP kinase signaling were added at 3 h post transfection. Data shown represent means ± SEM of triplicate assays. Percentages indicated by the same letter do not significantly differ (α = 0.05).

Discussion Despite the possible importance of alphaherpesvirus interactions with cellular signal transduction pathways, only few studies have addressed this issue. In a previous study, we described that the interaction between PRV envelope glycoprotein gD and axons of porcine TG neurons triggered small Rho GTPase and MAP kinase signaling and that intracellular signaling culminated in the formation of varicosities that may serve as virus exit sites for progeny virus particles (De Regge et al, 2006b). Addition of nectin-1 specific antibodies also induced this varicosity formation, suggesting that the trigger originated from the interaction between PRV-gD and one of its well described cellular receptors, nectin-1. Because signaling studies are cumbersome in primary neurons, a more straightforward in vitro model was used in this study to further characterise morphological changes and induction of signaling

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pathways associated with the interaction between gD and nectin-1. We analysed the effect of transfection of PRV-gD in CHO cells that either express (CHO-nectin-1) or lack (wt-CHO) nectin-1. We found that transfection of PRV-gD in monolayers of CHO-nectin-1 cells resulted in the formation of filopodia-like structures while these structures were only present in a minority of wt-CHO cells transfected with PRV-gD. This indicates that PRV-gD interaction with nectin-1 induces the formation of the filopodia-like structures. At first sight, this is in line with a recent report showing that nectin-1-dependent filopodia formation occurs in CHO cells during entry of HSV-1 (Clement et al, 2006). The number of transfected CHO-nectin-1 cells that showed filopodia-like structures was significantly higher after transfection of endocytosis deficient forms of PRV-gD compared to transfection of wt-gD. This indicates that the trigger resulting in formation of filopodia-like structures is probably given by a gD – nectin-1 interaction at the cell surface and not inside the transfected cells. Interestingly, after transfection of PRV-gD in CHO-nectin-1 cells, the formation of filopodia-like structures was only observed in the transfected cells and not in the cells contacting the gD expressing cell. This could be explained by at least two hypotheses. It might be that PRV-gD interacts with nectin-1 in cis (on the same cell) and that nectin-1 subsequently passes the trigger for the formation of filopodia-like structures to the cell. Another possibility is that gD interacts with nectin-1 present on contacting cells in trans. In this case, the signal is passed on to the cell via gD. It has been described before that HSV-gD can substitute for nectin-1 in adherens junctions of HSV infected or HSV-gD transfected cells and that gD can trans-interact with nectin-1 on a contacting cell (Krummenacher et al, 2003). This is thought to be a strategy of the virus to keep nectin-1 in the contacting cells at the cell contact sites to facilitate cell-to-cell spread. By performing co-seeding experiments, we showed that the induction of filopodia-like structures depends on a trans interaction of PRV-gD with nectin-1. Filopodia-like structures were still formed when PRV-gDtrunc was transfected in wt-CHO cells (eliminating interaction with nectin-1 in cis) and CHO-nectin-1 cells were added after transfection (providing nectin-1 in trans), but not vice versa. The number of transfected cells showing filopodia-like structures in these co-seeding experiments providing nectin-1 in trans was however lower than numbers observed after gDtrunc transfection in monolayers of CHO-nectin-1 cells. This can probably be explained by the fact that new cell-cell contact have to be established in this co-seeding experiment, whereas cell-cell contacts are already present when monolayers are transfected. Together with our observation that filopodia were only present in the gD expressing cells and

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not in the contacting cells, this indicates that the signal for formation of filopodia-like structures after interaction between gD and nectin-1 is transmitted to the cell via gD. This is different from our earlier observation on gD – nectin-1 mediated varicosity formation in TG neurons (De Regge et al, 2006b), where the signal is given to the cell via nectin-1. Thus, nectin-1 expressing CHO cells are not a good model to study signaling pathways underlying gD - nectin-1 mediated varicosity formation. How gD is able to transmit an intracellular signal is unclear at present. In this context, it is interesting that cells transfected with gDtrunc, a gD mutant that only expresses the first 5 amino acids of the cytoplasmic tail, also formed filopodia-like structures after interaction with nectin-1, indicating that the cytoplasmic tail of PRV-gD is probably not necessary to activate the signaling leading to the formation of filopodia-like structures. The cytoplasmic tail of gD has already been shown to be dispensable for several functions of gD. Entry of HSV-1 in host cells occurs as efficient in the absence of the cytoplasmic tail as when wt-gD is present (Browne et al, 2003; Cocchi et al, 2004; Jones & Geraghty, 2004) and the ectodomain of gD on itself has been shown to block HSV-1 induced apoptosis (Zhou et al, 2003). It might be that a conformational change occurs in the ectodomain of gD after interaction with nectin-1, as is proposed to occur after attachment of gD to its receptor during virus entry (Connolly et al, 2005), which could be sufficient on itself to initiate signaling in the cell or to allow gD to interact with another membrane protein that initiates signaling. In a previous study, we described that signaling resulting in formation of varicosities after interaction between PRV-gD and neurons of the trigeminal ganglion was dependent on small Rho GTPases and MAP kinases signaling (De Regge et al, 2006b). Although the initiation of signaling in CHO cells is clearly different, we found that gD mediated signaling induced by interaction between PRV-gD and nectin-1 after transfection of PRV-gD in CHO-nectin-1 cells also relies on these two major signaling pathways. We found that Rac1 and Cdc42 small Rho GTPases were involved in the formation of filopodia-like structures after transfection of endo--gD in CHO-nectin-1 cells. Interference with these two Rho GTPases by the use of specific inhibitors or by overexpressing dominant negative forms of Rac1 or Cdc42, resulted in a strong reduction in the number of gD transfected CHO-nectin-1 cells showing filopodialike structures. Inhibition of RhoA did not affected filopodia-like structure formation. This is in line with general knowledge that filopodia and lamellipodia formation relies on Rac1 and Cdc42 signaling, but not on signaling via RhoA (Hall, 1998). Further in line with these results, it has been shown that trans interactions between nectins induce formation of

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lamellipodia and filopodia in fibroblasts and epithelial cells which is also dependent on Rac1 and Cdc42 signaling (Kawakatsu et al, 2002). By making use of specific inhibitors of MAP kinases, we found that ERK-MAP kinase is involved in the formation of filopodia-like structures induced by the interaction between PRV-gD and nectin-1. Not much is known about a possible involvement of MAP kinases in the formation of filopodia. On the other hand, MAP kinase signaling induced by one or more proteins of PRV may not be unlikely since HSV-1 has also been described to activate MAP kinase signaling, i.e. via JNK, which seems to enhance virus replication (McLean & Bachenheimer, 1999; Galdiero et al, 2004; Diao et al, 2005b). Other reports on signaling induced by alphaherpesviruses indicate that HSV entry activates calcium signaling pathways (Cheshenko et al, 2003 and 2007) and that HSV-1 infection activates signaling leading to activation of NF-kappaB (Diao et al, 2005a; Hargett et al, 2006; Teresa Sciortino et al, 2007). Summarizing the signaling data of this current study and our previous study (De Regge et al, 2006b), we found that the interaction between PRV-gD and nectin-1 activates small Rho GTPase and MAP kinase signaling in two different in vitro model systems in which the interaction between gD and nectin-1 occurs at timepoints that represent two different phases of a normal alphaherpesvirus infection. The interaction between PRV-gD and the neurons occurs at the moment of attachment while transfection of CHO cells and subsequent expression of gD rather mimics the new formation of gD that normally occurs after virus has entered the cell. In both settings, signaling triggered by the interaction between PRV-gD and nectin-1 led to changes in cell morphology. An important difference is that during entry, gD – nectin-1 dependent signaling occurs via nectin-1 while signaling induced by the interaction of newly formed gD with nectin-1, at least in this transfection setup, occurs via gD. The observed induction of small Rho GTPase signaling in both models at timepoints that mimic different phases of an alphaherpesvirus infection could fit with the results described by Hoppe et al (2006) who showed that Rac1 and Cdc42 are activated a first time immediately after entry of HSV-1 in MDCK cells until 30 min post infection and a second time at 2 h post infection, a timepoint at which gD expression has already been shown to be detectable in KB epithelial cells (Cohen et al, 1980). Future studies will have to delineate whether the gD – nectin-1 induced filopodia-like structures are also formed during PRV infection to determine whether the induction of this process is not limited to the transfection setup and may or may not have biological

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significance. This research may be hampered by the fact that PRV has a growth defect on CHO cells (Nixdorf et al, 1999). Formation of filopodia after alphaherpesvirus infection has already been suggested to constitute a strategy of the virus to facilitate entry of extra virus particles by bringing them from the cell surrounding to the plasma membrane (Clement et al, 2006). On the other hand, the induced filopodia-like structures might be able to promote intracellular spread when they could bring virus in contact with surrounding non-infected cells. A sortlike mechanism to promote virus spread was already described for cell projections induced by PRV-US-3 (Favoreel et al, 2005).

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Chapter 5: Interferon alpha induces establishment of a latencylike state of alphaherpesvirus infection in trigeminal ganglion neurons in vitro

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_____________________________________Manuscript in preparation

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Abstract Alphaherpesviruses are important neurotropic viruses of man and animals, causing mild to severe disease symptoms, often related to the latency/reactivation cycle of the virus in trigeminal ganglion neurons. Little is known about the molecular mechanisms of this latency/reactivation cycle, at least in part due to the lack of a representative in vitro model to study latency. We report that addition of interferon alpha (IFN−α) to trigeminal ganglion neurons in a homologuous in vitro model that mimics the natural route of infection is sufficient to establish a stable but reactivatable latency-like state of infection of wild type pseudorabies virus (PRV, the swine alphaherpesvirus) in the majority of infected neurons. The latency-like state was sustained for several days after the withdrawal of IFN−α and a forskolin treatment resulted in reactivation of PRV in 50% of the infected neurons. Remarkably, IFN−α induced transition to the latency-like state was accompanied by rapid accumulation and subsequent dissappearance of IE180, the PRV orthologue of the HSV-1 immediate early protein ICP4. These data describe the first in vitro model in which a latency/reactivation cycle of a wild type alphaherpesvirus can be reconstituted using a physiologically relevant component of the immune system and point to a crucial role for IFN−α in the regulation of IE protein expression and in the establishment of alfaherpesvirus latency.

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Introduction Alphaherpesviruses are a subfamily of the herpesviruses containing closely related human and animal pathogens.

Herpes simplex virus 1 (HSV-1) is the prototype of human

alphaherpesviruses causing diseases ranging from mild labialis and stomatitis to blinding keratitis and in some rare cases to lethal encephalitis. Important animal alphaherpesviruses include the porcine pseudorabies virus (PRV) and bovine herpesvirus 1 (BoHV-1) causing respiratory symptoms, abortions and/or neurological symptoms. Primary infection of the host starts with a productive infection in epithelial cells of the upper respiratory tract, followed by virus entry in axons that innervate the infected mucosal surface (Baskerville, 1973). Then, the virus is transported retrogradely to sensory neuronal cell bodies in ganglia of the peripheral nervous system, with neurons of the trigeminal ganglion being the predominant target cells for HSV-1, PRV and BoHV-1 (Gutekunst et al, 1980; Ackermann et al, 1982; Croen et al, 1987). Mostly, a brief period of replication in the neurons is followed by the establishment of a latent infection in which functional viral genomes are retained in neuronal nuclei without virus production, causing a lifelong infection of the host (Roizman & Knipe, 2001). Specific stimuli such as immunosuppression can lead to periodic reactivation from this latent state which may result in new virus production and recurrent disease after anterograde axonal transport to the site of primary infection (Sainz et al, 2001). Although latency obviously is a critical aspect of the alphaherpesvirus lifecycle, many of the mechanisms resulting in latency and reactivation are not well understood (Preston, 2000). On the one hand, properties of neurons, being non-replicating terminally differentiated cells and the necessity for virus to travel long distances to reach the neuronal nucleus, are considered crucial for the fact that latent infections are induced in this cell type (Nichol et al, 1996; Kristie et al, 1999). On the other hand, results from many in vivo and in vitro studies indicate a critical importance of the hosts immune system during latency. From the earliest stage of infection, the virus is subject to control by the immune system. Primary infection at the epithelium and in the trigeminal ganglia is initially countered by the innate immunity. A well know action of the innate immune system is the production of type I interferons (IFN α/β) which induce the expression of several antiviral components e.g. double-stranded RNA dependent protein kinase R (PKR), 2’,5’-oligoadenylate synthetase (OAS), RNase L, Mx proteins and others, resulting in an antiviral state of the cell (reviewed in Levy & GarciaSastre, 2001). Other actions of the innate immunity are the nitric oxide and tumor necrosis

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factor α (TNF-α) production by macrophages, macrophage activation and early IFN-γ production by γδ TCR+ T cells and the action of NK cells (Liu et al, 1996; Kodukula et al, 1999). Notwithstanding the fact that alphaherpesviruses have developed several strategies to counter this antiviral response (Levy & Garcia-Sastre, 2001), the innate immune system plays a key role in controlling the acute phase of infection. During the first 5 to 7 days after infection, the adaptive immune response develops in lymph nodes and consists of production of B-cells secreting virus-specific neutralizing antibodies and proliferation of CD4+ helper and CD8+ cytotoxic T-cells (Cantin et al, 1995; Liu et al, 1996; Lang & Nikolich-Zugich, 2005). Several studies have shown the infiltration of virus-specific CD8+ T cells in the TG which are retained there in close association with neurons for long periods of time (Shimeld et al, 1995; Liu et al, 1996; Khanna et al, 2003; Theil et al, 2003; Van Lint et al, 2005; Verjans et al, 2007). This CD8+ T cell retention in the ganglia, together with the detection of some viral lytic gene transcripts and proteins in latently infected ganglia, have led different authors to suggest that there probably is a frequent or constant low level of virus reactivation in some neurons that is being repressed by the CD8+ T cells by a non-cytolytic mechanism, mainly by the production of IFN-γ (Liu et al, 2001; Khanna et al, 2003; Decman et al, 2005b). In this way, the virus is maintained in a latent state and reactivation is repressed. Hence, based on the data obtained during the last years, it is becoming generally accepted that the immune system plays an important yet far from fully understood role in establishing and maintaining alphaherpesvirus latency. Very little is known about the events initially leading to the establishment of latency in sensory neurons and the role of the immune system herein. In this study, we used a homologous in vitro two-chamber model consisting of primary porcine trigeminal ganglion neurons and the porcine alphaherpesvirus PRV to examine the capability of interferons to direct a wild type virus in a stable latency-like state of infection in its target neurons. We show that treatment of the neurons with IFN-α is sufficient to induce a latencylike state of infection that is sustained for several days in more than 60% of the infected neurons after the withdrawal of IFN-α. A subsequent forskolin treatment triggered 50% of the initially latently infected neurons to reactivate. This is the first report describing an in vitro system in which the cyclical pathway of infection, repression and reactivation is reconstituted using wild type virus and natural components of the immune system.

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Materials and methods Cultivation, virus inoculation, and analysis of primary TG neuronal cultures in a twochamber model Porcine trigeminal ganglion (TG) neurons were obtained as described before (Geenen et al, 2005) and seeded in an in vitro model, based on the ‘Campenot’ system (Campenot, 1977), that allows to simulate the in vivo route of neuronal infection (De Regge et al, 2006a,b). In brief, porcine trigeminal ganglia were excised from 4 to 6 week old piglets and dissociated by enzymatic digestion with 0.2% collagenase A (Roche, Mannheim, Germany). The harvested cells were resuspended in culture medium (MEM supplemented with 10% fetal bovine serum, 100U/ml penicillin, 0,1 mg/ml streptomycin, 0,1 mg/ml kanamycin and 30 ng/ml nerve growth factor (Sigma Chemical Company, St.Louis, MO, USA)) and seeded in the inner chamber of an in vitro two-chamber model. The two-chamber system consists of a polyallomer tube that is fixed with silicon grease on a collagen coated cover glass inserted in a 6-well plate (Nalge Nunc International, Rochester, NY, USA). The inside of the tube forms the inner chamber, the outside forms the outer chamber. The silicon barrier prevents diffusion of medium or virus from one chamber to the other (De Regge et al, 2006a). One day after seeding, cultures were washed with RPMI (Gibco BRL, Life Technologies Inc., Gaithersburg, MD) to remove non-adherent cells and from then on, culture medium was changed three times a week. After two to three weeks of cultivation, when clear axon growth could be observed in the outer chamber, two chamber models were ready for inoculation with PRV. All infections were done with wild type PRV strain Becker (Card et al, 1990). Two hours after inoculation of the outer chamber with 2×107 PFU of PRV, medium containing PRV was removed and the outer chamber was washed twice with culture medium. Afterwards new culture medium supplemented with polyclonal antibodies to PRV and guinea pig complement (Sigma-Aldrich) was added to prevent further continuous infection pressure from the outer chamber to neurons in the inner chamber. In experiments where interferons were used, both the inner and outer chamber were pretreated with interferon for 24h. Except for the presence of interferon, the infection was done as in control experiments. After removal of PRV from the outer chamber at 2hpi, interferon was no longer present in the outer chamber but remained present in the inner chamber for the total time of the experiment as indicated in the text.

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The ratio between viral antigen positive neurons in the inner chamber to the number of axons in the outer chamber after different treatments was determined by fluorescent labeling of neurons and viral antigens. For each condition, at least 20 axons with outgrowth in the outer chamber were examined. Data shown represent means ± SEM of triplicate assays. Antibodies, proteins and chemicals Polyclonal porcine FITC-labeled anti-PRV antibodies (Nauwynck and Pensaert, 1995b) were used to detect late PRV proteins, especially gB, gD and gE (Geenen et al, 2005). Polyclonal rabbit anti-IE180 antibodies were a gift of Prof. Tabares (Gomez-Sebastian and Tabares, 2004). The monoclonal neuronal marker mouse anti-neurofilament 68 was purchased from Sigma-Aldrich. Texas red-labeled goat anti-mouse antibodies and FITC-labeled goat antirabbit antibodies were obtained from Invitrogen. Recombinant porcine interferon alpha (PBL Biomedical Laboratories) and gamma (R&D systems) were used. Forskolin was obtained from Sigma-Aldrich. Immunofluorescence staining procedures After being washed in PBS, neuronal cultures in the inner and outer chamber of the twochamber model were fixed in 100% methanol for 20 min at -20°C. All antibodies were diluted in PBS, all to a dilution of 1:100. Cells were incubated with each antibody for 1h at 37°C and were washed two times in PBS in between all incubations steps and after the last incubation step. Confocal microscopy Stainings were analysed on a Leica TCS SP2 laser scanning spectrum confocal system (Leica Microsystems GmbH, Heidelberg, Germany) linked to a Leica DM IRBE microscope (Leica Microsystems GmbH). Images were taken using a 63x oil objective (NA 1.40-0.60) at room temperature and using Leica confocal acquisition software (Leica Microsystems GmbH). Adjustments of brightness and contrast were applied to the entire images and were done using Adobe Photoshop (Adobe Systems Inc., San Jose, CA).

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Results Interferon alpha and gamma suppress pseudorabies virus replication in TG neurons at 24h post infection Two-chamber models, consisting of TG neuronal cultures grown in an inner chamber with outgrowth of axons to an outer chamber separated from the inner chamber by a virus- and medium-impermeable silicon barrier (De Regge et al, 2006a,b), were used to study the antiviral effect of IFN−α and –γ on a PRV infection of TG neurons. In a first step, a quantification method was established that allows to detect a possible suppressing activity of interferons on productive virus replication. To this end, PRV infected two-chamber models were simultaneously stained with monoclonal anti-neurofilament antibodies (to visualize neurons) and polyclonal FITC-labeled anti-PRV antibodies (to visualize late PRV proteins, especially gB, gD and gE (Geenen et al, 2005)). When two-chamber models were analysed at 24h post infection (pi) with 2.107 plaque forming units (PFU) of PRV, the ratio between the number of viral antigen-positive neurons in the inner chamber to the number of axons grown through the silicon barrier into the outer compartment almost equaled one. This shows that for each axon that grows into the outer chamber, the corresponding neuron is productively infected in the inner chamber. This ratio can therefore be used to quantify the effect of interferon on productive viral replication in TG neurons. The effect of several doses of IFN-α and -γ at 24hpi was analysed (Figure 1). In these experiments, the inner and outer chamber of the two-chamber model were pretreated with either of the IFNs for 24h, followed by the addition of 2.107 PFU of PRV to the outer chamber. At 24hpi in the presence of interferons, the two-chamber models were fixed and processed as described above. Quantification showed that both interferons reduced productive virus infection in a dose-dependent manner with a reduction ranging from 64% at 0.5 U/ml to 98% reduction at 500 U/ml for IFN−α. Reduction with IFN−γ was less pronounced, ranging from 45% at 0.5 ng/ml to 81% at 50 ng/ml. These results show that interferons, especially IFN−α, are able to efficiently repress productive virus replication in TG neurons up to 24hpi.

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Figure 1. Interferon alpha and gamma suppress PRV replication in TG neurons at 24h post infection. Ratio between the number of late viral protein positive TG neurons in the inner chamber and the number of axons in the outer chamber in untreated, IFN-α (0.5; 5; 50; 500 U/ml) or IFN-γ (0.5; 50 ng/ml) treated, PRV infected two-chamber models at 24 h post infection. Data represent mean ± SEM of triplicate assays.

Interferon alpha suppresses pseudorabies virus replication in TG neurons for several days To address if the suppressive effect of IFN−α is sustained over a longer period of time, twochamber models were pretreated with 500 U/ml IFN−α for 24h, followed by inoculation with PRV in the outer chamber and fixation at 120hpi. IFN−α was present in the inner chamber during the entire experiment. After staining and analysis of the two-chamber models, 3 different stages of infection were observed (Figure 2 and 3).

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Figure 2. Productively PRV infected TG neurons and associated viral spread in the two-chamber model at 5 days post infection. Confocal images of TG neuronal cultures in the inner chamber of PRV-infected twochamber models at 5 days post infection in the presence of IFN-α (500 U/ml) and stained for neurofilament (Texas Red) and late viral antigens (FITC). Presence of late viral proteins in the neuron (A) and associated spread of newly produced infectious virus from the neuron to non-neuronal cells (B). (bar = 50µm)

Figure 3. Quantification of late viral protein expression in PRV infected TG neurons at 5 and 8 days post infection. Percentage of PRV infected TG neurons i) negative for late viral proteins, ii) Golgi-positive for late viral proteins or iii) Golgi-positive for late viral proteins with associated viral spread at 5 or 8 days post infection. Neurons grown in two-chamber models were treated with culture medium supplemented with IFN-α (500 U/ml) till 120 h post infection. Some models were analyzed at that timepoint, others at 192 h post infection after neurons were incubated with new culture medium or new culture medium supplemented with forskolin (200 µM) for 3 days. Data represent mean ± SEM of triplicate assays.

The vast majority (± 90%) of TG neurons showed no detectable expression of late viral antigens, indicating that the virus was in a repressed state in these neurons and did not lead to productive virus infection. In a small percentage of neurons (± 6%), late viral antigen expression was limited to the Golgi (see Geenen et al, 2005: co-localisation between late viral antigens and the cis-Golgi marker giantin) without spread of the virus to non-neuronal cells (Figure 2A). In an even smaller percentage of cells (± 4%), infected neurons were found in

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which new infectious virus particles were formed that had spread to the non-neuronal cells surrounding the cell body (Figure 2B). These results indicate that in the vast majority of TG neurons, IFN-α is able to suppress late viral antigen expression to undetectable levels up to 120hpi. The same experiment was conducted in the presence of 50 ng/ml IFN−γ. In this condition, all two-chamber models showed extensive virus spread in the inner chamber at 120hpi, indicating that the virus had overcome the antiviral state of the cells induced by IFN−γ (data not shown). These results indicate that IFN−α is able to efficiently suppress alphaherpesvirus replication for several days. Interferon alpha leads to IE180 protein shut-down in 55% of infected TG neurons at 120h post infection Previous results showed that IFN−α had a strong suppressive effect on the expression of late viral proteins in PRV infected TG neurons. In a next experiment, the effect of IFN−α on the immediate

early

(IE)

protein

expression

level

was

examined

by

performing

immunofluorescent stainings with antibodies directed to the only IE protein of PRV, i.e. IE180, and neuronal antigens. IE180 is a transcription factor essential to initiate viral replication and its HSV-1 homologue ICP4 is localised in discrete nuclear foci early during infection (de Bruyn Kops, 1998; Everett et al, 2004). In control infection experiments, without the use of IFN−α, IE180 expression localised in specific compartments in the nucleus could only be detected for a brief period early in infection, between 6 and 8 hpi (data not shown). In the presence of 500 U/ml IFN−α however, IE180 proteins, localised in discrete nuclear compartments (Figure 4), were detected in all infected neurons at 24hpi (Figure 5). At 120hpi in the presence of IFN−α however, localized nuclear IE180 expression was observed in only 45% of the infected neurons. The other 55% of infected neurons did not show any staining, indicating that these infected neurons had no detectable viral gene expression at that timepoint. These data indicate that all infected TG neurons proceed to expression of IE180 protein in the presence of IFN−α. However, within 5 days post inoculation, IE180 expression is reduced to undetectable levels in de majority of infected TG neurons.

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Figure 4. IE180 expression in distinct nuclear compartments in PRV infected TG neurons at 24 h post infection in the presence of IFN-α . Confocal image of a PRV-infected TG neuron in the inner chamber of a two-chamber model at 24 h post infection in the presence of IFN-α (500 U/ml) and stained for neurofilament (Texas Red) and IE180 (FITC). (bar = 20µm)

Figure 5. Quantification of IE180 expression in PRV infected TG neurons at 24 and 120h post infection. Ratio between the number of IE180 positive TG neurons in the inner chamber and the number of axons in the outer chamber in IFN-α (500 U/ml) treated, PRV infected two-chamber models at 24 and 120h post infection. Data represent mean ± SEM of triplicate assays.

The majority of infected TG neurons fail to proceed to late viral protein expression after withdrawal of IFN−α for 3 days Seen the strong suppressive effect of 500 U/ml IFN−α, abolishment of late viral gene expression in 90% of the infected neurons and repression of IE180 expression to undetectable levels in 55% of the infected neurons after 120hpi, the fate of infected neurons after the withdrawal of IFN−α was examined. Two-chamber models were pretreated with 500 U/ml

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IFN−α and infected with 2.107 PFU of PRV. At 120hpi, IFN−α was removed from the inner chamber by washing the cells. Two-chamber models were then further cultured for 3 days and finally fixed at 192hpi, followed by staining with antibodies to visualize neurons and late viral antigens. Quantification showed that after an incubation period of 3 days without the suppressing effect of IFN−α, 60% of the neurons still did not show detectable expression of late viral proteins (Figure 3), indicating that the virus is stably suppressed in these cells. When the same experiment was performed, and two-chamber models were stained for the presence of IE180 protein instead of late viral antigens, no infected neurons showing IE180 protein expression in discrete nuclear foci could be detected (data not shown). These results indicate that at 5 days post inoculation of TG neurons in the presence of IFN−α, the majority of neurons contains the virus in a stable dormant state. Addition of forskolin to neurons that contain virus in a stably suppressed state triggers them to spread the infection to neighbouring non-neuronal cells. To determine whether the IFN−α induced dormant state represents a true latency-like state of infection, we examined if the virus is able to reactivate from this dormant state. Two-chamber models were infected and treated with IFN−α as described before. At 120 hpi, IFN−α was washed away and medium supplemented with forskolin was added to the neurons. Forskolin increases cAMP levels in cells and is known to reactivate HSV-1 from quiescently infected primary neurons and neuronlike continuous cells in culture (Smith et al, 1992; Colgin et al, 2001; Danaher et al, 2003). Again, 3 days after replacement of the medium, two-chamber models were fixed, stained and analysed. Figure 3 shows that 3 days after forskolin treatment, 70% of the infected neurons showed expression of late viral proteins, compared to only 40% when medium without forskolin was added, and that infection spread from the vast majority of these neurons to neighbouring non-neuronal cells. This experiment shows that a forskolin treatment induces reactivation of PRV in 50% of the neurons that contained dormant virus at 120hpi. These reactivation data indicate that the IFN−α induced dormant state of the virus represents a state that closely mimics true viral latency.

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Discussion The ability of alphaherpesviruses to establish a latent infection in neurons of its host is a crucial aspect of the lifecycle of these viruses. It allows these viruses to remain present in the host for the entire lifetime and to reactivate in response to several stimuli, which can be associated with the production of new virus that can cause recurrent disease symptoms and can spread to new hosts. Many studies have been undertaken, both in vivo and in vitro, to unravel the mechanisms that lead to the induction and maintenance of this latent state of infection and resulted in the commonly accepted view that the latency/reactivation cycle is strictly controlled by a poorly understood interaction between virus, neurons and immune system (Divito et al, 2006). Many questions and doubts remain, especially since the latency/reactivaton cycle has up to date not been reconstituted using wild type virus, neurons and components of the immune system in vitro (Preston, 2000; Efstathiou & Preston, 2005). In this study, we used a homologous in vitro two-chamber model, based on the porcine alphaherpesvirus PRV and porcine TG neurons, that allows to mimic the natural route of infection to study the role of the immune system, more in particular of interferons, in the establishment of a latent infection. Both type I (IFN−α and –β) and type II (IFN−γ) interferons, as well as other cytokines like TNF−α, IL2, IL4, IL6, IL10 and NO producing cells have been reported to be present in TG of mice at the time that a latent infection is induced (Liu et al, 1996; Halford et al, 1997; Shimeld et al, 1997; Carr et al, 1998; Kodukula et al, 1999; Peng et al, 2005). Using our model, we found that IFN−α and –γ were able to strongly suppress virus replication at 24hpi at a point before ‘late’ viral protein expression (the FITC-labeled polyclonal anti-PRV serum used shows strong immuno-reactivity against gB, gD and gE (Geenen et al, 2005)), but IFN−α showed a stronger dose-dependent reduction in the number of late protein expressing neurons compared to IFN−γ. The suppressive effect of IFN−α was sustained for longer periods of time since 90% of the infected neurons still did not show detectable late viral gene expression at 120hpi. In contrast, the effect induced by IFN−γ was not able to maintain most of the infected neurons in a repressed state, seen by the extensive cytopathic effect in the inner chambers of all two-chamber models analysed at 120hpi. This indicates that IFN−γ alone is unlikely to be a decisive component to direct alphaherpesviruses in a latent state of infection during primary infection. During a herpesvirus infection, viral proteins are expressed in a sequential fashion after the genome has been transported to the nucleus. Late viral proteins are expressed late in infection

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and are accompanied by infectious virus production and virus spread. Immediate early proteins are the very first viral proteins expressed after infection and constitute transcription factors that initiate the subsequent transcription of early and late proteins. Without these immediate early proteins, viral replication does not initiate (Roizman & Knipe, 2001). PRV expresses only one protein with IE kinetics, IE180, the functional homologue of ICP4 of HSV-1 (Cheung et al, 1989; Martin et al, 1990; Wu & Wilcox, 1991). ICP4 is the first HSV-1 protein to be produced upon infection after the viral DNA associates with distinct cellular nuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10 (Liptak et al, 1996; Lukonis & Weller, 1997; Lukonis et al, 1997; de Bruyn Kops et al, 1998; Everett et al, 2003, 2004). The period that the ICP4 foci are localised juxtaposed to ND10 is very limited during lytic infection because upon ICP0 expression, another IE protein of HSV1, major components of the ND10 structures, Sp100 and PML, are degraded in a proteasome dependent fashion relying on ICP0, resulting in a disruption of these structures (Everett et al, 2000 and 2004; Boutell et al, 2002 and 2003). The PRV homologue of ICP0, i.e. EP0, has been shown to contain the zinc-binding RING finger motif necessary for this function and transfection assays showed that EP0 indeed could destabilise ND10 components leading to the disruption of these structures (Parkinson & Everett, 2000 and 2001). After disruption of ND10, further replication of alphaherpesviruses occurs in the nucleus in replication compartments that originate from some of the ICP4 foci after early gene expression starts and DNA replication begins (Knipe et al, 1987; Taylor et al, 2003; Everett et al, 2004). Several studies showed that IFNs can block HSV-1 and PRV infection at the level of IE gene or protein expression in primary mouse macrophages and several mice and human continuous cell lines (Mittnacht et al, 1988; Oberman & Panet, 1988 and 1989; De Stasio & Taylor, 1990; Nicholl and Preston, 1996; Tonomura et al, 1996; Pierce et al, 2005). In untreated TG neurons, we found that, as expected, PRV IE180 could be detected at low abundancy in discrete nuclear foci early in infection (6-8hpi). However, when our two-chamber model was infected in the presence of IFN−α, all infected neurons showed IE180 protein expression located in specific nuclear compartments at 24hpi. Because of the generally present, long lasting localised expression of IE180 in infected IFN−α treated neurons in comparison to the low abundant, short living localised expression in infected non-treated neurons, it is tempting to speculate that IFN−α blocks PRV replication after IE expression before the onset of early protein expression in TG neurons. This would confirm the results obtained in primary macrophages and continuous cell lines. It can however not be excluded that the observed

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localised compartments containing IE180 proteins in infected IFN−α treated neurons represent replication compartments, which could indicate that IFN−α imposes a block in viral replication after IE180 expression and before late viral protein production, probably at the point of DNA replication. Showing IE180 containing compartments juxtaposed to intact ND10 would provide evidence that EP0 is not expressed and would favor a block of IFN−α in the onset of EP0 expression. A lack of knowledge about the presence of ND10 in neurons however hinders this conclusion. Some studies reported that PML bodies were absent in neurons and other cells of neuronal lineage (Lam et al, 1995; Negorev and Maul, 2001), while a more recent study show the presence of PML bodies in human dorsal root ganglion neurons (Villagra et al, 2004). We were not able to detect ND10 in mock or IFN−α treated trigeminal ganglion neurons using anti-PML monoclonal antibodies (Sigma-Aldrich, data not shown). Although the hypothesis that IFN−α blocks viral replication between immediate early and early protein expression needs further investigation, our current results already reveal crucial information about the state of the viral genome in the IFN−α treated infected neurons. They show that IFN−α has no gross influence on the entry of PRV in the neuronal axonal endings and subsequent retrograde spread to the nucleus and prove that all infected neurons received a functional viral genome. Interestingly, at 120hpi in the presence of IFN−α, only 45% of the infected neurons showed detectable IE180 expression. This shows that 55% of the infected neurons do no longer show any detectable viral gene expression after a 5 days incubation period with IFN−α, and since latently infected neurons per definition do not show any viral gene expression (Roizman & Knipe, 2001) raises the possibility that the viral genome was directed in a latency-like state in these neurons. After removal of medium containing IFN−α from the inner chamber at 120hpi and replacing it by medium without any suppressive component, 60% of the infected neurons did not proceed to detectable expression of late viral proteins within the next 3 days, indicating that the virus is stably suppressed in these cells. Of possible importance is the correlation between the number of neurons that do not express late viral antigens 3 days after withdrawl of IFN−α at 120hpi (60%) and the number of neurons that no longer showed detectable IE180 expression at 120hpi (55%). This leads to the hypothesis that specifically those neurons that still express IE180 at 120hpi proceed to expression of late viral proteins when the suppressive stimulus is withdrawn. This is supported by the observation that at 3 days after IFN−α was removed at 120hpi, no IE180 expression in distinct nuclear compartments could be detected in any neuron. This leads us to speculate that a shut-down of IE180 protein expression is

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necessary to install a stably suppressed state of viral infection in neurons and that the presence of IE proteins suffices to proceed to productive infection upon removal of the suppressive agent. An important aspect in the definition of a true latent infection is the possibility of the virus to reactivate from the latent state. Reactivation in vivo is mostly observed after physical or psychological stress or in immunocompromised patients and is correlated with a suppression of the immune status of the host (Sainz et al, 2001). The exact molecular mechanism leading to reactivation is not yet known. Several stimuli have been shown to lead to reactivation of HSV-1 from latently infected mice in vivo and from quiescently infected cells in vitro (Smith et al, 1992; Millhouse & Wigdahl, 2000; Arthur et al, 2001; Colgin et al, 2001; Danaher et al, 2003 and 2005; Hunsperger & Wilcox, 2003ab; Neumann et al, 2007). One of these methods is the treatment of the cells with forskolin, a molecule which increases cAMP levels in the cell. We found that forskolin induced reactivation of PRV in our two-chamber model in 50% of neurons containing stably suppressed virus at 120hpi, since it induced spread of the infection from these neurons to the neighbouring non-neuronal cells. This confirms that the stably suppressed virus infection in TG neurons can be reactivated and therefore can be considered to represent a state that closely mimics true viral latency. It will be interesting to test whether neurons that contain the virus in a stably suppressed state in our in vitro system express latency associated transcripts (LATs). Although several reports indicate that many latently infected neurons do not express detectable levels of LATs (Ramakrishnan et al, 1994 and 1996; Mehta et al, 1995, Maggioncalda et al, 1996, Wang et al, 2005), arguing against the use of LAT expression as a mark for latency, expression of these transcripts in the absence of other viral transcripts is still widely considered as a molecular hallmark of herpesvirus latency (Roizman & Knipe, 2001). In future studies, we will therefore develop an in situ hybridisation assay to test whether LATs are expressed in the in vitro system described here. A definition of latency at the molecular level is difficult to formulate seen the contradictory data about possible gene expression during a latent infection (Roizman & Knipe, 2001; Decman et al, 2005a). A more classical definition that makes sense at the clinical level could be the following: a latent herpesvirus infection in vivo is defined as the presence of a functional wild type viral genome in neurons of the host without the production of new virus particles. Upon specific stimuli, the virus can reactivate and spread to other cells, which may cause recurrent disease symptoms. The results discussed above show that PRV infection of TG neurons in our two-chamber model in the presence of IFN−α fullfill all criteria mentioned

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in the definition and represents the first induction of a latency-like state of infection in vitro that closely mimics latency in vivo and that makes use of a wild type alphaherpesvirus and physiologically relevant components of the immune system. With this in vitro model in which a natural latency-like state of alphaherpesvirus infection can be induced, we provide a promising tool to dissect the molecular details of alphaherpesvirus latency and reactivation. Besides its usefullness in fundamental research, several possible application purposes are situated in the field of drug development. Till now, very little drugs are available to treat recurrent symptoms associated with reactivation of alphaherpesviruses, all of them being derivates of acyclovir (Woo & Challacombe, 2007). Our model could be a valuable tool to identify components that interfere with the latency/reactivation cycle, thereby being possible candidates for curative treatment of recurrent disease symptoms. For about 20 years, HSV has been studied as a promising vector to deliver transgenes to neurons, in this way helping to cure neuronal diseases (Broberg & Hukkanen, 2005). Our model could be a helpfull tool to screen the capacity of recombinant alphaherpesviruses designed for neuronal gene therapy to go into a latent state of infection under physiological relevant conditions and their capacity for high and long lasting expression of the transgene. Several implications of the discussed results may add to the knowledge about establishment of latency. The fact that all neurons proceed to late viral protein expression upon infection in the absence of IFN−α show that the virus-neuron interaction on itself is not sufficient to induce and maintain a latent infection, further supporting the view that the host immune system is a third necessary partner needed in this process (Divito et al, 2006). IFN−α appears to be able to act as an important component of the immune system in the establishment of a latent alphaherpesvirus infection, by preventing late protein expression and driving virus in a stably suppressed state in a majority of neurons. Our results suggest that a suppression of virus protein expression at the IE level may be required to prevent an immediate progress to the lytic cycle of infection when suppressive stimuli disappear. Several hypotheses explain why IFN−α on itself induces a latency-like infection in 60% but not in all infected neurons in the two-chamber model. A longer incubation time with IFN−α may function to suppress viral protein production in the remaining neurons or the presence of one or more extra pro-inflamatory cytokines may be needed to establish latency in a subset of TG neurons. The latter seems acceptable since other cytokines than IFN−α have been detected in the TG of mice at the time that a latent infection is induced (Liu et al, 1996;

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Halford et al, 1997; Shimeld et al, 1997; Carr et al, 1998; Kodukula et al, 1999; Peng et al, 2005). More research is needed to clarify this. In conclusion, we show that addition of IFN−α to neurons of the trigeminal ganglion in vitro is sufficient to drive wild type PRV in a stable but reactivatable latency-like state of infection in a majority of infected TG neurons. This is the first physiologically relevant in vitro model that

closely

mimics

the

latency/reactivation

cycle

of

alphaherpesviruses.

__________________________________________________________________________6.

Chapter 6: General discussion

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____

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Neurons of the trigeminal ganglion are very important for several alphaherpesviruses including herpes simplex virus 1, bovine herpesvirus 1 and porcine pseudorabies virus. They constitute major target cells for virus spread and lifelong latent infections and are critical for the recurrent and neurological aspects of disease associated with these viruses. Disease symptoms associated with HSV-1, the prototype of human alphaherpesviruses, range from mild labiales and stomatitis to blinding keratitis and in rare cases to lethal encephalitis. Animal alphaherpesviruses such as BoHV-1 and PRV can cause respiratory symptoms, abortions and neurological symptoms. Upon infection of a host, primary replication of most alphaherpesviruses occurs in epithelial cells of the upper respiratory tract. Newly produced virions that could evade the innate immune response at the place of initial infection subsequently enter the axon endings of trigeminal ganglion neurons that innervate these epithelial cells. Entry of HSV and PRV in axons of these sensory neurons is thought to be initiated by interaction between the viral envelope protein gD with its receptor nectin-1 (Haarr et al, 2001; Mata et al, 2001; Milne et al, 2001; Richart et al, 2003; Ono et al, 2006). This interaction triggers a fusion between the viral envelope and the axolemma which is mediated by viral proteins gB, gD, gH and gL and results in the release of capsids and tegument into the cytoplasm. This is followed by retrograde transport of these capsids and a part of the associated tegument to the nucleus by means of microtubule-associated fast axonal transport. Once the viral genome has entered the nucleus, either a lytic infection with the production of new virions is started, or a latent infection is established. The choice between those two infection patterns in not well understood and probably reflects the fine balance that exists between the viral properties promoting a lytic infection and the suppressive environment favoring latent infection created by the neurons and the immune system (Preston, 2000). Newly produced virions, during primary infection or after reactivation from the latent state, are transported back to the periphery by antrograde axonal transport. It is not clear how new virions are transported in the anterograde direction, either as completely assembled enveloped virions or as subvirion particles in which the capsid and tegument are transported separately from glycoprotein containing vesicles (Cunningham et al, 2006). Transport of virions is followed by the release of virus at the axon terminus where spread to surrounding cells can cause recurrent disease symptoms and spread to new hosts. At the periphery, the virus is subject to the action of both the innate and adaptive immune response that will finally clear the virus from the infected peripheral sites. Besides the spread at the axon termini, it has also been observed that some cells along the axon shaft of the neuron become infected after PRV infection of the rat retina,

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indicating that the virus can also exit the axon along the axon shaft (Ohara et al, 2001; Tomishima & Enquist, 2002). PRV infection of TG neurons induces functional varicosities which may serve as virus exit sites Although the interactions between alphaherpesviruses and TG neurons are of critical importance for the latency/reactivation cycle and recurrent viral spread, many of these interactions are far from fully understood. A major problem for the study of interactions between alphaherpesviruses and TG neurons is the lack of easy-to-handle, relevant model systems. Therefore, we developed a homologous in vitro model that mimics the in vivo situation as close as possible. The developed in vitro two-chamber model is based on the use of porcine TG neurons and PRV. Because of the significant homology between PRV and HSV-1, PRV can be used as a model for this human alphaherpesvirus for many aspects of its lifecycle (Pomeranz et al, 2005). Our model is based on a model described by Campenot (1977) and consists of a polyallomer tube that is fixed with silicon grease on a collagen coated cover glass. Control experiments showed that the silicon barrier is medium- and virus impermeable. Dissociated TG cells were seeded in the inner chamber. After 2 to 3 weeks of cultivation, outgrowth of newly formed axons through the silicon barrier could be observed. When clear axon growth was observed in the outer chamber, two-chamber models were infected by adding PRV to the outer chamber. Confocal analysis of stainings revealed that at 24 hpi, multiple neuronal cell bodies in the inner chamber showed expression of PRV antigens whereas non-neuronal cells did not express viral antigens at that timepoint. Thus, PRV enters the inner chamber via neuronal axons that are present in the outer chamber and reaches the neuronal cell body by retrograde axonal transport where a productive infection is started. This illustrates the capacity of the model to mimic the natural route of infection. At 24 hpi, the ratio between the number of viral antigen-positive neurons in the inner chamber to the number of axons grown through the silicon barrier into the outer chamber was determined for numerous two-chamber models and virtually equaled one. This indicates that for each axon that grows into the outer chamber, the corresponding neuron in the inner chamber becomes productively infected. This is in further support of the view that the interaction between alphaherpesviruses and TG neurons on itself is not sufficient to induce a

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latent infection and that additional factors, most likely the host’s immune system, are needed in this process. Analysis of stained two-chamber models at 24 hpi led to the surprising observation that more than 70% of axons of PRV-infected neurons showed a massive number of bouton-like axonal swellings. These swellings were only detected on 12% of axons of non-infected neurons. This indicated that the interaction between PRV and axons of TG neurons triggered the formation of the swellings. The observation that synaptophysin, a protein that is present on synaptic vesicles that can contain neurotransmitters, accumulated specifically in these swellings identified them as varicosities, also known as synaptic boutons (Pannese, 1994). The induction of varicosities upon PRV interaction with axons of TG neurons is a novel aspect of the cell biology of alphaherpesvirus interaction with neurons and it represents the first description of a virus infection that stimulates varicosity formation. From 24 hpi onwards, it was found that virus spreads from the TG neuron to neighbouring non-neuronal cells, both from the cell body and along the axon shaft. Presence of infected non-neuronal cells along the axon shaft has been observed before in in vivo studies (Kritas et al, 1994ab and 1995; Ohara et al, 2001; Shimeld et al, 2001; Tomishima & Enquist, 2002). Interestingly, a large majority (88%) of single-infected non-neuronal cells along the axon was located juxtaposed to varicosities, indicating that egress of infectious virus along the axon shaft occurs specifically at varicosities. In this context, it is interesting that a study of Tomishima & Enquist (2002) described that after PRV infection of the rat retina, PRV egress occured along the axon shaft of optic nerve axons at scattered sites. Recently it was also found in vitro that axonal egress of PRV occurs at scattered sites (Ch’ng & Enquist, 2005). Given our current finding that axonal egress almost exclusively occurs at varicosities, we hypothesize that these scattered sites may correspond to varicosities. This is further supported by the finding that growth cones and varicosities might constitute assembly places for HSV-1 and serve as axonal exit places along human fetal dorsal root ganglia neurons in vitro (Saksena et al, 2006). We also found that the spread of PRV from varicosities to non-neuronal cells could not be blocked by neutralizing antibodies, thereby showing that it occurs via direct cell-to-cell spread. This is in line with the finding of Tomishima & Enquist (2002) that a gDnull mutant, which is not infectious as extracellular virus but is capable of spreading by cell-cell contacts, could infect glial cells at scattered sites along the axon shaft. Both existing models of anterograde virus transport, the traditional and the subvirion model (cfr. 1.3.4), can fit with virus exit at varicosities. On one hand, secretory vesicles which are believed to transport enveloped virus particles in the traditional model have been shown to accumulate at

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varicosities and on the other hand, varicosities could serve as assembly places of mature virus particles in the subvirion model because they have been shown to constitute functional synapses, just like axon termini (Santos et al, 2001). Our finding that PRV induces varicosities indicates that the virus may actively increase the number of axonal exit sites for progeny virus. Although future in vivo experiments will be required to confirm varicosity formation in vivo by alphaherpesvirus interaction with TG neurons, and to delineate the biological significance of such an observed induction, different speculations can be put forward how varicosity induction could promote viral spread in vivo. i) It has been shown that axonal end regions of TG neurons that pass the basement membrane are prone to varicosity formation, most likely because of the absence of a myelin sheath once they have crossed this barrier. Therefore, varicosity formation could facilitate virus spread from terminal axon regions to epithelial cells at the periphery, a crucial step in alphaherpesvirus pathogenesis (Kondo et al, 1992; Ibuki et al, 1996). ii) The induction of varicosities along the axon shaft could also lead to egress of virus along the axon shaft to surrounding cells. Based on observations of in vivo studies however, the biological relevance of this would be unsure, since glial cells have been shown not to support lytic virus production, thereby inhibiting further viral spread (Townsed & Collins, 1986; Wilkinson et al, 1999). iii) It is possible that varicosity formation may also influence transneuronal spread, i.e. the spread of virus from one neuron to another across synaptic contacts, certainly when the induced varicosities would be able to form synaptic contacts with other neurons (which is a known property of certain varicosities (Hu et al, 2004b)). The property of transneuronal spread of alphaherpesviruses has been used extensively to map neuronal circuits (Enquist, 2002; Song et al, 2005). Taken together, the virally induced varicosity formation along axons of TG neurons could be a newly described strategy of alphaherpesviruses to modulate the host cell in order to facilitate virus spread. Other examples of such modulation have been reported before, e.g. the gE mediated targeting of virus particles to cell junctions to promote spread in polarized cells (Collins and Johnson, 2003; Wisner & Johnson, 2004) and the promotion of intercellular spread via US3-induced actin rearrangements and cell projections (Favoreel et al, 2005). PRV induced varicosities were further characterized and were found to be functional varicosities that had an intact synaptic transmission. This was based on their capacity to internalise FM1-43, a fluorescent marker for firing neurons (Mizoguchi et al, 2002). Despite the fact that we did not clarify if varicosities synapsed with adjacent non-neuronal cells, the

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intact synaptic transmission should enable them to release neurotransmitters, either to the contacting cell or otherwise directly in the extrasynaptic space (Zhu et al, 1986; Glanzman et al, 1989; Pannese, 1994; Hatada et al, 1999, Kim et al, 2003; Vizi et al, 2004). In this context, it is interesting to note that alphaherpesvirus infections have been associated with hyperexcitability of neurons, which may be involved in acquired epilepsy after HSV-1 encephalitis (Chen et al, 2004a). Further investigations will have to unravel whether the PRVinduced formation of synaptically effective varicosities lead to changes in excitability of neurons. Although speculative, the possible release of neurotransmitters from induced varicosities might also be interesting in the context of the severe neurological symptoms observed after PRV infection of young piglets which are absent in older animals. A morphological difference between post-natal and older piglets that might influence the disease symptoms upon PRV infection is the state of the myelinisation of the nervous system. It is known that the nervous system is not completely matured at birth and that the myelinisation process of both central and peripheral nervous system continues till adolescence. Several studies concerning the development of the central nervous system also showed that unmyelinated axons are prone for varicosity formation before the onset of myelinisation, a property that disappears afterwards (De Neef et al, 1982; Hartman et al, 1982; Rozeik & Schulz-Harder, 1990; Rodier, 1995; Fernandez & Nicholls, 1998; Morgan, 2001; Berthold & Nilsson, 2002). In vivo studies examining the maturation and myelinisation of the peripheral nervous system and the formation of varicosities in post-natal piglets upon PRV infection will be able to clarify if varicosity induction, and possible associated release of neurotransmitters, in unmyelinated axons has an impact on the observed disease symptoms. PRV-induced varicosities depend on gD, nectin-1, Rho GTPase and MAP kinase signaling How does PRV induce varicosities? An important first step towards answering this question was to specify during which step of the PRV infection cycle the trigger for varicosity formation is given. The finding that UV-inactivated PRV could induce varicosity formation, together with the fact that varicosities were already present at 6 hpi, showed that the trigger was given during virus attachment or entry, before the onset of viral protein production. A PRV mutant that lacks the gB envelope protein, and therefore is still able to attach to but not enter cells, was still able to induce varicosities, indicating that attachment of the virus is sufficient for varicosity induction. Interestingly, a PRV mutant that lacks the gD envelope protein, which also still can attach to but not enter cells, could not induce varicosity formation

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in TG neurons. This showed that PRV-gD is necessary in this process. Addition of soluble recombinant PRV-gD to the axons induced varicosity formation in a dose-dependent manner, showing that PRV-gD in not only necessary but also sufficient for varicosity induction. This is in line with observations of Mizoguchi et al (2002) who showed that addition of recombinant HSV-gD to mouse hippocampus neurons, as a trigger to induce nectin-1 signaling, resulted in a substantial increase in the number of synaptophysin-positive varicosities. PRV-gD interacts with sensory neurons by binding to its receptor. Nectin-1 is supposed to be the PRV receptor on sensory neurons (Haarr et al, 2001; Mata et al, 2001; Milne et al, 2001; Richart et al, 2003; Ono et al, 2006). This was indirectly supported by the observation that addition of nectin-1 specific antibodies to our two-chamber model, as a surrogate ligand instead of gD, also triggered varicosity formation. However, it can not be ruled out that other nectins or nectin-like molecules might be relevant to the gD-mediated induction of varicosities because no direct evidence was provided for an interaction between PRV-gD and nectin-1 on TG sensory neurons in our system. Together, these data indicate that interaction of PRV envelope protein gD with its receptor on axons of TG neurons, probably nectin-1, during virus attachment provides the trigger necessary for subsequent formation of varicosities. Our data may also be important with regard to a newly developing field in virus research: signaling pathways induced by the interaction between alphaherpesvirus envelope proteins and their receptors during virus entry. Addition of peptides of glycoprotein H of HSV to the outside of cells has been shown to activate JNK-MAP kinase signaling (Galdiero et al, 2004). Recently, signaling induced by binding of HSV-gD to another known entry receptor, HVEM, has been reported to induce signaling that leads to activation of NFkappaB (Teresa Sciortino et al, 2007). Because little is known about the signaling started by interaction of gD with nectin-1, we examined which signaling pathways are involved in the formation of varicosities induced by the interaction between gD and TG neurons, probably via nectin-1. Inhibitor studies were performed to check the involvement of small Rho GTPases and MAP kinase signaling pathways. It was found that the formation of varicosities in TG neurons induced by the interaction between PRV-gD and axons at the moment of virus attachment was dependent on signaling via Cdc42 small Rho GTPase and p38 MAP kinase. This is in line with results showing that formation of varicosities in sensory neurons of Aplysia is dependent on signaling via Cdc42 and MAP kinases (Hu et al, 2004a; Udo et al, 2005). Also studies in a C. elegans

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model system showed that p38 MAP kinase signaling is involved in varicosity formation (Nakata et al, 2005). Interestingly, interactions between nectins have been shown to be important for the formation of synapses and this synapse formation is hypothesized to rely on Cdc42 signaling (Takai et al, 2003b), because it has been shown that nectins signal via Rac1 and Cdc42 small Rho GTPases for several processes such as formation of adherens junctions and lamellipodia and filopodia formation (Kawakatsu et al, 2002; Fukuhara et al, 2003). gD-transfected nectin-1-expressing CHO cells are not a good model to study gD – nectin-1 dependent varicosity formation in TG neurons A more extensive examination of signaling pathways induced by the interaction between PRV-gD and its receptor on neurons was difficult because several drawbacks are associated with the use of in vitro cultures of primary TG neurons to study signal transduction at the molecular level. They are cumbersome and time-consuming, primary neurons can not be transfected with standard transfection protocols thereby leaving transduction as the only option to transiently express foreign proteins in neurons, and the presence of the non-neuronal cells invariably leads to strong background signals in Western blotting experiments. Furthermore, there is no absolute guarantee that the signaling leading to formation of varicosities is triggered by an interaction between gD and nectin-1, also other nectins or nectin-like molecules might be involved. Therefore, we examined whether the interaction between PRV-gD and nectin-1 does result in intracellular signaling leading to morphological changes in another, more straightforward model system in which the effect of transfection of PRV-gD in CHO cells that either express or lack nectin-1 was analysed. It was found that transfection of PRV-gD in CHO-nectin-1 cells induced formation of filopodia-like structures and this change in cell morphology appeared to be dependent on the interaction between gD and nectin-1. Co-seeding experiments indicated that it was especially the interaction between PRV-gD on the transfected cell and nectin-1 on the contacting cells in trans that triggered the formation of the filopodia-like structures. Trans interactions between newly synthesized gD after HSV infection or after transfection of HSV-gD in nectin-1 expressing melanoma cells with nectin-1 have been shown before (Krummenacher et al, 2003) and they appear to be a strategy of the virus to keep nectin-1 in the contacting cells localised at the junctions with the infected cell, in this way facilitating cell-to-cell spread. Future studies will have to delineate whether the filopodia-like structures are also formed during PRV infection of CHO-nectin-1 cells to

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determine any biological relevance of these findings. Formation of filopodia after alphaherpesvirus infection has already been suggested to constitute a strategy of the virus to facilitate entry of extra virus particles by bringing them from the cell surrounding to the plasma membrane (Clement et al, 2006). On the other hand, the induced filopodia-like structures might be able to promote intracellular spread when they could bring virus in contact with surrounding non-infected cells. A sortlike mechanism to promote virus spread was already described for cell projections induced by PRV-US-3 (Favoreel et al, 2005). Interestingly, filopodia-like structures were only observed on the transfected cells and not on contacting cells. Together with the aforementioned finding that it is the gD interaction with nectin-1 in trans that triggers the formation of the filopodia-like structures, this indicates the the trigger for filopodia formation is passed on to the cell via gD and not via nectin-1. This constitutes a new aspect of gD – nectin-1 signaling and is different from our findings on gD induced varicosity formation where the trigger is passed on to the neuron via nectin. Interestingly, transfection of a truncated form of PRV-gD, that almost lacks the entire cytoplasmic domain, in CHO-nectin-1 cells also induced formation of filopodia-like structures, indicating that the trigger passed on to the cell through gD is independent of the cytoplasmic tail of gD. The cytoplasmic tail of gD has already been shown to be dispensable for several functions of this protein. Entry of HSV-1 in host cells occurs as efficient in the absence of the cytoplasmic tail as when WT-gD is present (Browne et al, 2003; Cocchi et al, 2004; Jones & Geraghty, 2004) and the ectodomain of gD on itself has been shown to block HSV-1 induced apoptosis (Zhou et al, 2003). It might be that a conformational change occurs in the ectodomain of gD after interaction with nectin-1, as is proposed to occur after attachment of gD to its receptor during virus entry (Connolly et al, 2005), which could be sufficient on itself to initiate signaling in the cell or to allow gD to interact with another membrane protein that initiates signaling. Our earlier studies in neurons indicated that specific Rho GTPase and MAP kinase signaling pathways are involved in nectin-mediated signaling during gD-nectin interactions. We went on to determine if and which Rho GTPase and MAP kinase dependent pathways could also be involved in gD-mediated signaling during gD – nectin-1 interactions after transfection of gD in CHO-nectin-1 cells. Inhibitor studies showed that the formation of filopodia-like structures was dependent on signaling via Rac1 and Cdc42 small Rho GTPases and ERK-MAP kinase. The involvement of Rac1 and Cdc42 small Rho GTPases was confirmed by co-transfections of PRV-gD and dominant negative forms of Rac1 or Cdc42 in CHO-nectin-1 cells. These

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results are in line with studies showing that filopodia and lamellipodia formation relies on Rac1 and Cdc42 signaling, but not on signaling via RhoA (Hall, 1998). Further in line with these results, it has been shown that trans interactions between nectins induce formation of lamellipodia and filopodia in fibroblasts and epithelial cells which is also dependent on Rac1 and Cdc42 signaling (Kawakatsu et al, 2002). Less is known about the role of MAP kinases in the formation of filopodia, although the Rho GTPase and MAPkinase signaling pathways appear to be intimately interwoven (Bagrodia et al, 1995; Coso et al, 1995; Juliano et al, 2004). On the other hand, MAP kinase signaling induced by one or more proteins of PRV may not be unlikely since HSV-1 has also been described to activate MAP kinase signaling, i.e. via JNK, what seems to enhance virus replication (McLean & Bachenheimer, 1999; Galdiero et al, 2004; Diao et al, 2005b). Summarizing the signal transduction data, it appears that the interaction between PRV-gD and nectin-1 can induce morphological changes in different cell types at two time points that may represent different phases of a normal alphaherpesvirus infection. The interaction between PRV-gD and probably nectin-1 in the neuronal model that induces formation of varicosities occurs at the moment of attachment while transfection of gD in CHO cells that leads to formation of filopodia-like structures is a model that mimics the new formation of gD after virus has entered the cell. However, the initiation of the signaling cascade is very different in the two systems used. Indeed, a very important difference is that the signal in the neuronal model is probably passed on to the cell via nectin-1 whereas gD appears to pass the signal in the CHO model. Despite the differences between both models, in both cases, interaction between PRV-gD and nectin-1 appears to trigger similar Rho GTPase and MAP kinase signal transduction pathways. Both models include Cdc42 and/or Rac1 Rho GTPase signaling. This could fit with results of Hoppe et al (2006) who showed that Rac1 and Cdc42 are activated a first time immediatly after entry of HSV-1 in MDCK cells until 30 min post infection and a second time at 2 h post infection, a timepoint at which gD expression has already been shown to be detectable in KB epithelial cells (Cohen et al, 1980). A novel in vitro model to simulate alphaherpesvirus latency A very important interaction between alphaherpesviruses and TG neurons is the lifelong latency/reactivation cycle. It allows the virus to remain present in the host for the entire lifetime and to reactivate in response to several stimuli, which can be associated with the production of new virus particles that can cause recurrent disease symptoms and spread to

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new hosts. The latency/reactivation cycle is divided in three phases: establishment, maintenance and reactivation. There are strong indications that the interaction between virus – neuron – immune system is crucial during each phase of the latency/reactivation cycle but many molecular mechanisms underlying the three phases are still unclear (Divito et al, 2006). An involvement of the innate immune system in the establishment of latency has been put forward based on reports on the presence of several immune cell types and cytokines associated with the innate immune response in the TG of infected mice at the time that a latent infection is induced (Liu et al, 1996; Halford et al, 1997; Shimeld et al, 1997; Carr et al, 1998; Kodukula et al, 1999; Peng et al, 2005). These immune cells and cytokines include macrophages, γδ-Tcells, NK cells, type I and II IFNs, TNF−α, IL-2, IL-4, IL-10 and NO producing cells. Interference with one or some of these components often resulted in a decreased establishment of latency and mice that died from encephalitis (Halford et al, 1997 and 2006; Sciammas et al, 1997; Kodukula et al, 1999; Leib et al, 1999; Minami et al, 2002). This indirectly indicates that these factors may be involved in efficiently suppressing the infection and driving the virus into a latent state. Because IFNs are present in the TG at the timepoint that a latent infection is installed and since IFNs are unusual in that they are able to directly act on virus replication, the effect of IFNs on productive PRV replication in neurons was evaluated in our in vitro two-chamber model. Because non-neuronal cells are not sites of latency for HSV-1 and PRV in the ganglion, the exclusive infection of neurons in our two-chamber model is an extra advantage to investigate the capacity of IFNs to induce latency. Both IFN−α and –γ were able to suppress virus replication in neurons at 24 hpi at a point before late viral protein expression, but IFN−α showed a stronger dose-dependent reduction in the number of late protein expressing neurons compared to IFN−γ. The suppressive effect of IFN−α was sustained for longer periods of time since 90% of infected neurons still did not show detectable late viral gene expression at 5 dpi. In contrast, IFN−γ was unable to maintain the infected neurons in a repressed state, seen the extensive cytopathic effect in the inner chambers of all two-chamber models analyzed at 5 dpi. This indicates that IFN−γ alone is unlikely to be the decisive component to direct alphaherpesviruses in a latent state of infection. When IFN−α was removed from the inner chamber at 5 dpi and replaced by medium without any suppressive component, still 60% of the infected neurons did not proceed to detectable expression of late viral proteins within the next 3 days, indicating that the virus is stably suppressed in this subpopulation of neurons. The presence of the stably

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repressed virus led to the hypothesis that this virus was possibly directed into a latency-like state in the neurons by IFN−α. Several premises however have to be fullfiled before it can be concluded that a latent PRV infection is installed by the addition of this natural component of the immune system to our in vitro model. i) Neurons must harbour functional wild type PRV, ii) no viral proteins are expressed during the latent state of infection and iii) it must be possible to reactivate the virus from the latent state. Stainings of infected, IFN−α treated two-chamber models fixed at 24 hpi revealed that all infected neurons expressed IE180 in discrete nuclear compartments at that timepoint. Untreated TG neurons only showed such discrete IE180 foci early in infection and invariably proceed to later stages of infection, with undetectable IE180 at 24 hpi. Because of the generally present, long lasting localised expression of IE180 in infected IFN−α treated neurons in comparison to the low-abundant, short living localised expression in infected nontreated neurons, it is tempting to speculate that IFN−α blocks PRV replication at the point of IE180 expression before the onset of early protein expression in TG neurons. This would be in line with studies that showed that IFNs can block HSV-1 and PRV replication at the level of IE gene or protein expression in primary mouse macrophages and in several mice and human continuous cell lines (Mittnacht et al, 1988; Oberman & Panet, 1988 and 1989; De Stasio & Taylor, 1990; Nicholl & Preston, 1996; Tonomura et al, 1996; Pierce et al, 2005). It can however not be excluded that the observed localised expression of IE180 represents an early rather than an immediate stage in infection. Indeed, the observed IE180 foci may represent replication compartments since it has been shown that ICP4 may localize in these structures (Knipe et al, 1987; de Bruyn Kops et al, 1998). In this case, IFN−α would block viral replication after IE180 expression and before late viral protein production. Future studies examining a possible colocalisation between IE180 and viral early proteins known to be present in replication compartments will help to clarify this issue. Nevertheless, the present data reveal crucial information about the state of the viral genome in the IFN−α treated neurons. They show that IFN−α has no gross influence on the entry of PRV in the neuronal axonal endings and subsequent retrograde spread to the nucleus. The detectable expression of IE180 in all infected neurons also prove that all these neurons received a functional viral genome, thereby fullfiling the first premise for a latent infection. Interestingly, at 5 days post infection in the presence of IFN−α, only 45% of the infected neurons still had detectable IE180 expression. This shows that 55% of the infected neurons had shut down IE180 between 1 and 5 days of incubation with IFN−α. This subpopulation of

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neurons fullfills the second premise of latently infected cells and raises the possibility that the viral genome is present in a latent state in these neurons. An interesting question concerning this observation is: what causes the IE180 protein expression in neurons to fade away during the 5 days incubation period with IFN−α? Gradual degradation of VP16 originating from incoming virus could perhaps hamper the formation of an efficient transcription initiation complex, leading to a stop in IE promoter activition, associated with a complete block in IE180 protein expression. Alternatively, maybe IFN−α induced proteins involved in gene silencing or proteins present in ND10 need some time to completely silence the viral genome. Activity of proteases and/or the proteasome perhaps may also be involved in the IE180 shutdown. These are only a few hypotheses. It will be a challenging task for future studies to provide an answer to this question. When IFN−α was removed at 5 dpi, 40% of the neurons proceeded to late viral protein expression within 3 days. There is a correlation between the number of neurons that showed detectable IE180 expression at 5 dpi (45%) and the number of neurons that did proceed to late viral protein expression within 3 days after the removal of IFN−α (40%) and, although speculative, this led us to hypothesize that specifically those neurons that show detectable IE180 expression at 5 dpi proceed to later stages of infection, whereas the others contain virus in a stably suppressed conformation. Several hypotheses may explain why IFN−α can suppress all viral protein expression in a subpopulation of 55% of the infected neurons but not in all infected neurons in the two-chamber model. The kinetics of IE180 clearance by IFN−α was not examined in this study so it is possible that some neurons need a longer incubation time with IFN−α then 5 days to shut down IE180 expression. Another explanation can be found in the observed presence of several other cytokines than IFN−α in the TG at the time of latency establishment as mentioned before. This could indicate that some neurons need the stimulus of additional pro-inflammatory cytokines to efficiently suppress IE180 expression. These hypotheses will be evaluated by future studies. Addition of medium containing forskolin, a product that interacts with the cAMP signaling in the cell and has been shown to induce reactivation in several in vitro models of latency (Hunsperger & Wilcox, 2003a; Danaher et al, 2005), to the neuronal cultures after IFN−α was removed at 120 hpi induced reactivation of PRV in 50% of the neurons that contained stably suppressed virus within 3 days, since it induced spread of the infection from these neurons to neighbouring non-neuronal cells. This also fullfills the last premise, leading to the conclusion

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that IFN−α alone is able to direct PRV in a latency-like state in a subpopulation of TG neurons in vitro. In conclusion, the data discussed above show that addition of IFN−α to neurons of the trigeminal gangion in vitro is sufficient to drive wild type PRV in a stable but reactivatable latency-like state of infection in a majority of infected neurons in vitro. However, this does not exclude a possible involvement of other cytokines and immunological factors in efficient establishment of latency in vitro. The results described above show that the study of the latency/reactivation cycle in our twochamber model has already contributed to the knowledge about the establishment of latent infections. Because it enables us to induce a natural latent alphaherpesvirus infection in TG neurons, it will also be an excellent tool to dissect molecular details of the different phases of the latency/reactivation cycle, for example to investigate the order of events that lead to new virus production upon reactivation. Besides its usefullness in fundamental research, several potential application purposes may be situated in the field of drugs discovery. For example, it could be used to screen components for their capacity to maintain alphaherpesviruses in a latent state when stimuli that normally cause reactivation are applied. In this way possible candidates for curative treatment of alphaherpesvirus infections could be identified. Our model could also be a helpful tool to screen the capacity of recombinant alphaherpesviruses designed for neuronal gene therapy to go into a latent state of infection under physiological relevant conditions and their capacity for high and long lasting expression of the transgene. Conclusion In conclusion, the homologous in vitro two-chamber model promises to be an excellent tool to study the interaction between alphaherpesviruses and neurons of the trigeminal ganglion. Several till hereto unknown aspects of this interaction have been found and were described in this thesis. Many more remain to be discovered. However, despite the model’s unique properties to closely mimic the in vivo situation, it has to be kept in mind that these results obtained in vitro have to be verified in vivo to estimate their true relevance.

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Chapter 7: Summary – Samenvatting

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Summary Neurons of the trigeminal ganglion (TG) are very important target cells for several alphaherpesviruses. They constitute major target cells for virus spread and lifelong latent infections, two important aspect of the alphaherpesvirus lifecycle, both related with recurrent disease symptoms observed after reactivation. The aim of the current study was to investigate the interaction between the porcine alphaherpesvirus pseudorabies virus and porcine TG neurons in vitro in more detail with special emphasis for aspects of neuronal spread and latency. In Chapter 1, a brief introduction is given on PRV and TG neurons. Additionally, this chapter reviews the course of a lytic alphaherpesvirus infection of neurons and summerizes the current knowledge about the interaction between virus, neurons and immune system during the different phases of a latent infection. In Chapter 2, a homologous in vitro two-chamber model, based on the “Campenot” system, was developed to study the interaction between alphaherpesviruses and TG neurons. The model is based on the use of primary porcine TG neurons and the porcine alphaherpesvirus PRV. The two-chamber model consists of a polyallomer tube that is fixed with silicon grease on a collagen-coated cover glass inserted in a 6 well plate. The inside of the tube forms the inner chamber and the outside the outer chamber, separated from each other by the mediumand virus-impermeable silicon grease barrier. Primary porcine trigeminal ganglion neurons were obtained as described by Geenen et al (2005). Trigeminal ganglia were excised from 4 to 6 week old piglets and enzymatically and mechanically digested. The obtained cell suspension, consisting out neurons and non-neuronal cells, was seeded in the inner chamber of the two-chamber model. After 2 to 3 weeks of cultivation, outgrowth of newly formed axons through the silicon barrier could be observed by light microscopy and this was confirmed by immunofluorescent stainings visualising the neurons with a neuronal marker. It was demonstrated that addition of PRV to the outer chamber resulted in the presence of late viral antigen positive neurons in the inner chamber at 24 hpi whereas non-neuronal cells were not infected at that timepoint. At 48 hpi, cytopathic effect associated with PRV infection was observed in non-neuronal cells under the light microscope. The presence of late viral antigen positive non-neuronal cells observed after immunofluorescent stainings confirmed that PRV

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had spread from the neurons to the non-neuronal cells. It is concluded that the developed twochamber model allowed infection of TG neurons by PRV after retrograde axonal transport of the virus to the neuronal cell body, thereby mimicking the in vivo route of infection. In Chapter 3, the observation that the majority (> 70%) of axons of infected neurons showed a massive number of bouton-like swellings was studied in more detail. These swelling were only observed in 12% of non-infected neurons, indicating that interaction of PRV with the neurons triggered the swelling formation. In a first step, the nature of the swellings was determined. The observed accumulation of synaptophysin, a protein present in synaptic vesicles, in the swellings and their capacity to internalise FM1-43, a marker for firing neurons, led to the conclusion that the swellings represent functional varicosities. The induction of varicosities on neurons during an alphaherpesvirus infection is a complete new aspect of the cell biology of alphaherpesvirus – neuron interactions. It was never reported before that a virus induced varicosity formation during infection. It was found that infected non-neuronal cells were present along the axons of infected neurons. Almost each singleinfected non-neuronal cell (88%) was juxtaposed to a varicosity, indicating that the virus could use these structures to exit from the axon shaft. Addition of neutralizing antibodies to the neuronal cultures could not block the spread of the virus to the non-neuronal cells, indicating that the virus uses direct cell-to-cell spread to reach these cells. In a next step, it was determined during which phase of the infection cycle the trigger was given that culminated in the formation of varicosities. The fact that the varicosities were already present at 6 hpi and that UV-inactivated PRV also could induce varicosties indicated that the trigger was given early during infection, probably during virus attachment or entry. This was examined by making use of virus mutants genotypically and phenotypically negative for one of the glycoproteins known to be involved in attachment and entry. Addition of a PRV-gBnull mutant, that can attach to but can not enter cells, to the outer chamber resulted in the formation of varicosities, indicating that attachment of the virus to the axon is important for varicosity induction. In a next step, PRV-gDnull virus, which also still can attach to but not enter cells, was added to the outer chamber and also this virus could induce varicosity formation, further indicating that attachment to the axon via gD is important in this process. To further examine the role of gD in varicosity formation, soluble recombinant porcine gD was added to the outer chamber in a next experiment. It was found that also this soluble gD was capable to induce varicosities in a dose-dependent manner, showing that gD is not only

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necessary but also sufficient for the induction of varicosities. It is known that gD is important for attachment of the virus to the cell and that it starts the fusion process between the viral envelope and the cellular plasmamembrane after binding to its receptor. Several classes of gD receptors have been described, but several studies indicate that nectin-1 is the PRV-gD receptor on sensory neurons. This was indirectly confirmed by the observation that addition of antibodies directed to an epitope in the ectodomain of nectin-1, as an surrogate ligand instead of gD, also induced the formation of varicosities in a dose-dependent manner. These results show that the binding of PRV-gD with its neuronal receptor in the early steps of infection provides a trigger resulting in the formation of varicosities, structures that can be used by the virus to enhance its spread later during infection. In the last part of this study, the involvement of two signal transduction pathways in the induction of varicosities after gD-receptor binding was examined. Previous studies on signaling via nectins and signaling involved in the induction of varicosities had highlighted the importance of small Rho GTPase and MAP kinase signaling. Therefore, inhibitor studies were performed to verify if these signaling pathways were also important in varicosity induction in our model. Specific inhibition of Rho and Rac small Rho GTPases and ERK and JNK MAP kinases had no effect on the induction of varicosities. A broad range inhibitor of small Rho GTPases and specific inhibitors of Cdc42 small Rho GTPase and p38 MAP kinase signaling however suppressed varicosity formation to the same low level as observed in mock-infected cultures, indicating that the virus-induced varicosity formation in TG neurons depends on Cdc42 small Rho GTPase and p38 MAP kinase signaling. In chapter 4, the PRV-gD interaction with nectin-1 was studied in a more straightforward in vitro model system by comparing the effect of PRV-gD transfection in CHO cells that either express or lack nectin-1. It was found that transfection of vectors encoding endocytosis negative forms of PRV-gD in nectin-1 expressing CHO cells resulted in the formation of filopodia-like structures in about 38% of these cells, while this was only observed in about 10% of PRV-gD transfected CHO-cells lacking nectin-1. This indicates that the interaction between PRV-gD and nectin-1 can induce formation of filopodia-like structures in CHO cells. Transfection of WT-PRV-gD in CHO-nectin-1 cells also resulted in formation of filopodialike structures, but to a lesser extent (21%). This could be explained by the fact that it is known that WT-gD is efficiently internalised from the plasma membrane of the cell by endocytosis. Subsequently, this suggests that the interaction between PRV-gD and nectin that results in the formation of filopodia-like structures occurs at the cell surface. Co-seeding

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experiments showed that the induction of filopodia-like structures is dependent on an interaction between gD and nectin-1 present on contacting cells in trans. Together with the observation that the filopodia-like structures are only present on the transfected cells and not on the surrounding cells contacting it, this suggests that the signal to initiate the formation of these structures after the interaction between gD and nectin-1 was given to the cell via gD. Because also transfection of a truncated form of PRV-gD, that lacks the cytoplasmic domain except for the first 5 amino acids, in CHO-nectin-1 cells result in the formation of filopodialike structures, it appears that the cytoplasmic tail of gD is dispensable to pass the signal to the cell. In a next part of this study, it was examined whether the two signal transduction pathways, via small Rho GTPases and MAP kinases, which have been shown to be involved in signaling leading to the formation of varicosities after interaction of PRV-gD with TG neurons, were also involved in the formation of the filopodia-like structures. By using specific inhibitors of small Rho GTPases and MAP kinases and performing co-transfections with PRV-gD and dominant negative forms of Rac1 and Cdc42, it was shown that signaling via Rac1 and Cdc42 small Rho GTPases and ERK-MAP kinase is involved in the formation of the filopodia-like structures. It is concluded that transfection of PRV-gD in CHO-nectin-1 cells, a model that mimics the new formation of gD after infection of these cells with PRV, triggers intracellular signaling dependent on Rac1 and Cdc42 small Rho GTPases and ERKMAP kinase after an interaction between PRV-gD and nectin-1 in trans that leads to the formation of filopodia-like structures. In chapter 5, the effect of interferons on an alphaherpesvirus infection of neurons was studied in our two-chamber model. It was found that both IFN−α and IFN−γ had a suppressive effect on productive viral replication at 24 hpi but IFN−α showed a stronger dose-dependent reduction in the number of late viral antigen expressing neurons compared to IFN−γ. Furthermore, the suppressive effect of IFN−α (500 U/ml) on productive viral replication was sustained for long periods of time. At 5 days post infection, still 90% of the infected neurons showed no detectable late viral gene expression. In contrast, all neuronal cultures treated with IFN−γ showed extensive cytopathic effect at that timepoint, indicating that IFN−γ has not the ability to suppress productive PRV infection for 5 days. When IFN−α was removed from the medium at 5 days post infection and cells were cultured for 3 more days in medium without any suppressive agent, still 60% of the infected neurons showed no late viral protein expression, indicating that the virus is stably suppressed in those neurons. To get a better

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understanding of the stage of infection at which PRV is blocked by IFN−α, immunofluorescent stainings were performed to look at the presence of IE180, the PRV protein that is normally first expressed during lytic infection. Stainings of infected but untreated two-chamber models revealed that IE180 could be observed in discrete nuclear compartments in infected neurons between 6 and 8 hpi but never at later time points. In contrast, all infected neurons in IFN−α (500U/ml) treated cultures showed the localised IE180 expression in the nucleus at 24 hpi. This shows that IFN−α blocks virus replication at a point after IE180 expression and before late viral protein expression. The presence of IE180 in all infected neurons also indicates that all neurons received a functional PRV genome. Interestingly, at 5 days post infection in de presence of IFN−α, only 45% of the infected neurons still had detectable IE180 expression. This observation shows that IFN−α suppressed IE180 protein expression in the other 55% of infected neurons during the 5 days incubation period, implicating that those neurons had no longer any detectable viral protein expression. In a next experiment, the reactivatability of the stably suppressed viral genomes was evaluated. It was demonstrated that addition of forskolin, a product that interferes with cAMP signaling in the cell and is known to induce reactivation in several in vitro models of latency, to the medium after a 5 days incubation period with IFN−α resulted in the reactivation of PRV in 50% of neurons containing stably suppressed virus within a 3 day time period, since it induced spread of the infection from these neurons to the neighbouring non-neuronal cells. Together, these data show that the stably suppressed genomes fullfill all criteria that are present in the definition of a latent infection and therefore, it can be concluded that addition of IFN−α to TG neurons in vitro is sufficient to drive wild type PRV in a stable but reactivatable latency-like state of infection in a majority of infected TG neurons. This is the first physiologically relevant reconstitution of the latency/reactivation cycle of alphaherpesviruses in vitro. Two general conclusions come from this study about the interaction between PRV and trigeminal ganglion neurons in vitro: 1) PRV-gD interaction with its receptor on porcine trigeminal ganglion neurons triggers small Rho GTPase and MAPK kinase signaling. The activation of signal transduction pathways leads to varicosity formation. These virally induced varicosities early during infection (at the point of virus attachment) can subsequently be used as axonal exit sites by the virus to enhance its spread.

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2) Interferon alpha is a decisive factor in the establishment of a reactivatable latent wild type PRV infection in a subset of porcine trigeminal ganglion neurons in vitro. It suppresses viral protein expression at a point between immediate early and late viral protein expression at 24 hpi and clears all detectable protein expression in a majority of infected neurons at 5 dpi.

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Samenvatting Neuronen van het trigeminaal ganglion (TG) zijn zeer belangrijk voor verscheidene alfaherpesvirussen. Ze zijn van belang voor het spreiden van het virus en bovendien zijn het de voornaamste doelwitcellen voor de levenslange latentie/reactivatie cyclus. Dit zijn twee belangrijke aspecten in de levencyclus van alfaherpesvirussen, beide gerelateerd met de recurrente ziektesymptomen die waargenomen worden na reactivatie van het virus. De doelstelling van deze studie was om de interactie tussen het alfaherpesvirus van het varken, pseudorabies virus (PRV), en trigeminale varkensneuronen in vitro meer in detail te bestuderen, en dit met de nadruk op aspecten van het neuronaal spreiden en latentie. In hoofdstuk 1 wordt een introductie gegeven over PRV en TG neuronen. Tevens wordt het verloop van een lytische alfaherpesvirus infectie in neuronen behandeld en vervolgens wordt ook de huidige kennis over de interactie tussen virus, neuronen en het immuun systeem tijdens de verschillende fasen van latentie samengevat. In hoofdstuk 2 wordt beschreven hoe een homoloog in vitro twee-kamer model, gebaseerd op het ‘Campenot’ model, werd ontwikkeld dat kan gebruikt worden bij het onderzoek naar de interactie tussen alfaherpesvirussen en TG neuronen. Het model is gebaseerd op het gebruik van primaire varkensneuronen en het alfaherpesvirus van het varken PRV. Het twee-kamer model bestaat uit een poly-allomeer buisje dat met behulp van silicone op een met collageen gecoat draagglaasje is bevestigd dat in een 6 well plaat ligt. De ruimte binnen het buisje vormt de binnenste kamer en de ruimte buiten het buisje de buitenste kamer. Beide ruimtes zijn van elkaar gescheiden door de medium- en virus ondoorlaatbare silicone barrière. De primaire trigeminale neuronen van het varken werden bekomen op de manier die reeds werd beschreven door Geenen et al (2005). De trigeminale ganglia werden verwijderd uit 4 tot 6 weken oude biggen en vervolgens enzymatisch en mechanisch gedigesteerd. De bekomen celsuspensie die zowel uit neuronen als niet-neuronale cellen bestaat, werd dan in de binnenste kamer van het twee-kamer model geplant. Nadat we deze cellen 2 tot 3 weken gecultiveerd hadden konden nieuw uitgegroeide axonen die doorheen de silicone barrière naar de buitenste kamer waren gegroeid, waargenomen worden onder de lichtmicroscoop. Deze uitgroei van axonen naar de buitenste kamer werd tevens bevestigd via immunofluorescentie kleuringen met een neuronale merker om de neuronen te visualiseren. Er werd aangetoond dat

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het toevoegen van PRV aan de buitenste kamer resulteerde in neuronen die virale eiwitten bevatten in de binnenste kamer op 24h na infectie. De niet-neuronale cellen waren op dit tijdstip niet geïnfecteerd. Op 48h na infectie kon het cytopathisch effect dat geassocieerd is met een PRV infectie waargenomen worden in de niet-neuronale cellen onder de lichtmicroscoop. De aanwezigheid van viraal antigeen positieve niet-neuronale cellen op immunofluorescentie kleuringen bevestigde dat PRV vanuit het neuron naar de niet-neuronale cellen was gespreid. Uit deze studie konden we besluiten dat het mogelijk is om met behulp van het ontwikkelde twee-kamer model TG neuronen te infecteren met PRV na retrograad axonaal transport van het virus naar het neuronale cellichaam. Op deze manier wordt de in vivo infectieroute nagebootst. In hoofstuk 3 werd de vaststelling dat de meerderheid (> 70%) van axonen van geïnfecteerde neuronen blaasjes-achtige zwellingen vertoont in meer detail onderzocht. Deze zwellingen waren maar aanwezig op 12% van niet-geïnfecteerde neuronen wat aanduidt dat de interactie tussen PRV en de neuronen de vorming van de zwellingen veroorzaakt. In een eerste stap werd de natuur van deze zwellingen bepaald. De waargenomen opstapeling van synaptophysin, een eiwit dat aanwezig is in synaptische vesikels, en hun capaciteit om FM143 te internaliseren leidde tot de conclusie dat de zwellingen functionele ‘varicosities’ waren. De inductie van ‘varicosities’ op neuronen gedurende een alfaherpesvirus infectie is een nieuw aspect van de celbiologie van alfaherpesvirus – neuron interactie. Het werd nooit eerder gerapporteerd dat een virus infectie de vorming van ‘varicosities’ induceert. De vaststelling dat geïnfecteerde niet-neuronale cellen aanwezig waren langs de axonen van geïnfecteerde neuronen toonde aan dat PRV het axon niet enkel kan verlaten aan het axonuiteinde maar ook langs de axonschacht. Bijna elke niet-neuronale cel (88%) die afzonderlijk geïnfecteerd was, was vlak naast een ‘varicosity’ gelegen. Dit toonde aan dat het virus deze structuren kan gebruiken om de axonschacht te verlaten. Toevoeging van neutraliserende antistoffen aan de neuronale culturen kon de spreiding van het virus naar de niet-neuronale cellen niet verhinderen wat aantoont dat het virus deze cellen infecteert via direct cel-tot-cel spreiden. In een volgende stap werd bepaald tijdens welke fase van infectie de trigger werd gegeven die aanleiding geeft tot vorming van ‘varicosities’. Het feit dat de ‘varicosities’ reeds aanwezig waren op 6h na infectie en dat UV-geïnactiveerd PRV ook in staat was om de vorming van ‘varicosities’ te induceren duidde erop dat de trigger vroeg tijdens infectie wordt gegeven,

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waarschijnlijk tijdens aanhechting of binnenkomst van het virus. Dit werd verder onderzocht door gebruik te maken van PRV mutanten die zowel geno- als fenotypisch negatief waren voor bepaalde envelopeiwitten die van belang zijn tijdens aanhechting en binnenkomst van virus in de cel. Wanneer een gBnull mutant, die enkel nog kan aanhechten aan de cel maar niet meer kan binnendringen in de cel, werd toegevoegd aan de buitenste kamer, bleek deze in staat om de vorming van ‘varicosities’ te induceren. Dit was een aanwijzing dat virus aanhechting van belang is in de inductie van ‘varicosities’. In een volgende stap werd gDnullPRV, dat eveneens kan aanhechten aan de cel maar niet kan binnendringen in de cel, toegevoegd aan de buitenste kamer en dit resulteerde slechts in een inductie van ‘varicosities’ tot hetzelfde niveau als vastgesteld in controle experimenten. Dit toont aan dat aanhechting van virus aan neuronen via gD van belang is in de inductie van ‘varicosities’. Om het belang van gD verder te onderzoeken werd in een volgend experiment recombinant varkens gD toegevoegd aan de buitenste kamer. Ook dit recombinante gD bleek in staat om de ‘varicosities’ te induceren op een dosis-afhankelijke manier. Dit toont aan dat gD niet enkel nodig maar ook voldoende is voor inductie van ‘varicosity’ vorming. Het is gekend dat gD van belang is voor aanhechting van het virus aan de cel en dat het het fusie proces tussen de virale envelop en de cellulaire plasmamembraan op gang brengt na binding op zijn receptor. Verschillende klassen van PRV and HSV-1 receptoren zijn reeds beschreven, maar verschillende studies tonen aan dat nectine-1 waarschijnlijk de receptor is voor gD op sensorische neuronen. Dit werd in deze studie indirect bevestigd door de vaststelling dat toevoegen van antistoffen die gericht zijn tegen een epitoop in het ectodomain van nectine-1, als een vervangende ligand voor gD, ook de vorming van ‘varicosities’ induceren op een dosis-afhankelijke manier. Deze resultaten tonen aan dat de binding van PRV-gD op zijn neuronale receptor tijdens vroege stappen van infectie een trigger veroorzaakt die resulteert in de vorming van ‘varicosities’, structuren die door het virus kunnen gebruikt worden om het spreiden te bevorderen tijdens latere stappen van infectie. In een laatste deel van die studie werd de betrokkenheid van twee signaaltransductiewegen in de inductie van ‘varicosities’ na binding van gD op zijn receptor onderzocht. In studies die signaaltransductie via nectines en signaaltransductie die leidt tot vorming van ‘varicosities’ hadden onderzocht, werd reeds aangetoond dat signalisatie via small Rho GTPasen en MAP kinasen van belang is voor deze processen. Daarom werden studies met inhibitoren van deze pathways uitgevoerd om het belang ervan te onderzoeken in de inductie van ‘varicosities’ in ons model. Specifieke inhibitie van Rho en Rac small Rho GTPasen en van ERK en JNK MAP kinasen had geen effect op de inductie van ‘varicosities’. Een algemene inhibitor van

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small Rho GTPasen en specifieke inhibitoren van Cdc42 small Rho GTPase en p38 MAP kinase onderdrukten daarentegen wel de vorming van ‘varicosities’ en dit tot op hetzelfde niveau van mock-geïnfecteerde culturen. Dit toont aan dat de virus geinduceerde vorming van ‘varicosities’ in TG neuronen afhankelijk is van Cdc42 small Rho GTPase en van p38 MAP kinase. In hoofdstuk 4 werd de interactie tussen PRV-gD en nectine-1 bestudeerd in een ander, gemakkelijker manipuleerbaar, in vitro model. De proefopzet bestond in het vergelijken van het effect van PRV-gD transfectie in CHO cellen die al dan niet nectine-1 tot expressie brengen. Transfectie van expressievectoren die mutante vormen van gD tot expressie brengen die geen endocytose kunnen ondergaan in CHO-nectine-1 cellen resulteerde in de vorming van filopodia-achtige structuren in ongeveer 38% van de getransfecteerde cellen terwijl transfectie van PRV-gD in CHO-cellen zonder nectine-1 maar in ongeveer 10% van deze cellen resulteerde in vorming van filopodia-achtige structuren. Dit toont aan dat de interactie tussen PRV-gD en nectine-1 de vorming van filopodia-achtige structuren kan induceren in CHO cellen. Ook de transfectie van wt-PRV-gD in CHO-nectine-1 cellen resulteerde in de vorming van filopodia-achtige structuren. Deze structuren waren weliswaar in een lager aantal getransfecteerde cellen (21%) aanwezig in vergelijking met transfectie met endocytose mutante vormen van gD. Dit kan mogelijks verklaard worden door het feit dat gekend is dat wt-gD op de plasmamembraan gemakkelijk geinternaliseerd wordt via endocytose. De lagere inductie van filopodia-achtige structuren door wt-gD suggereert bijgevolg dat de interactie tussen gD en nectine-1 gebeurt aan het oppervlak van de cel. Co-seeding experimenten toonden aan dat de inductie van de filopodia-achtige structuren afhankelijk is van een trans interactie tussen gD op de getransfecteerde cel en nectine-1 aanwezig op cellen in contact met de getransfecteerde cel. Samen met de vaststelling dat de filopodia-achtige structuren enkel aanwezig zijn in de getransfecteerde cel en niet in de omgevende cellen, suggereert dit dat het signaal dat de vorming van deze structuren start na de interactie tussen gD en nectine-1 doorgegeven wordt naar de cel via gD. Aangezien ook transfectie van een getrunceerde vorm van PRV-gD, die de cytoplasmatische staart op uitzondering van de eerste 5 aminozuren mist, in CHO-nectine-1 cellen de vorming van de filopodia-achtige structuren induceert, lijkt het erop dat de cytoplasmatische staart van gD overbodig is om het signaal door te geven naar de cel.

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In een volgend deel van deze studie werd nagegaan of de twee signaal transductiewegen, nl. small Rho GTPasen en MAP kinasen, die betrokken waren bij de vorming van ‘varicosities’ na interactie van PRV-gD met TG neuronen ook van belang zijn voor de vorming van de filopodia-achtige structuren. Door gebruik te maken van specifieke inhibitoren voor de verschillende small Rho GTPasen en MAP kinasen en door co-transfecties uit te voeren met vectoren die coderen voor PRV-gD en voor dominant negatieve vormen van ofwel Rac1 of Cdc42 werd aangetoond dat signaaltransductie via Rac1 en Cdc42 small Rho GTPasen en via ERK-MAP kinase betrokken is bij de vorming van de filopodia-achtige structuren. We konden besluiten dat transfectie van PRV-gD in CHO-nectine-1 cellen, een model dat de nieuwvorming van gD na PRV infectie van deze cellen simuleert, intracellulaire signalisatie kon triggeren die afhankelijk is van Rac1 en Cdc42 small Rho GTPasen en ERK-MAP kinase na de interactie tussen PRV-gD en nectine-1. De geïnduceerde signalisatie leidt uiteindelijk tot de vorming van filopodia-achtige structuren op de getransfecteerde cel. In hoofdstuk 5 werd het effect van interferonen op een alfaherpesvirus infectie van neuronen in ons twee-kamersysteem onderzocht. Zowel IFN−α als IFN−γ bleken een onderdrukkend effect te hebben op productieve virus replicatie op 24 h na infectie maar de dosisafhankelijke reductie in het aantal neuronen die late virale eiwitten tot expressie brengen was sterker voor IFN−α dan voor IFN−γ. Het onderdrukkend effect van IFN−α (500 U/ml) op productieve virus infectie bleek bovendien ook langer aan te houden dan 24h. Op 5 dagen na infectie vertoonde 90% van de geïnfecteerde neuronen nog steeds geen detecteerbare late virale eiwitexpressie. Alle neuronale culturen die behandeld waren met IFN−γ daarentegen vertoonden een uitgebreid cytopatisch effect op 5 dagen na infectie. Dit toont aan dat IFN−γ niet de eigenschappen bezit om een productieve infectie gedurende een zo lange tijdspanne te onderdrukken. Wanneer IFN−α op 5 dagen na infectie uit het medium verwijderd werd en de cellen vanaf dan gedurende 3 dagen gecultiveerd werden in medium zonder verdere supplementen, bleek dat nog altijd 60% van de geïnfecteerde neuronen geen late virale eiwitexpressie vertoonde. Dit wijst erop dat het virus stabiel onderdrukt is in deze neuronen. In een volgend experiment werden immunofluorescentie kleuringen uitgevoerd om de aanwezigheid van IE180, het PRV eiwit dat normaal als eerste tot expressie komt tijdens lytische infectie, te onderzoeken in neuronen in de aan- of afwezigheid van IFN−α in het medium. Het doel hiervan was om een beter inzicht te verwerven over het tijdstip waarop IFN−α de productieve virusvermeerdering stillegt. Kleuringen van geïnfecteerde twee-kamer

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systemen die niet behandeld waren met IFN−α toonden aan dat IE180 kon gedetecteerd worden in geïnfecteerde neuronen tussen 6 en 8 h na infectie en dit gelocaliseerd in specifieke nucleaire compartimenten. In IFN−α (500 U/ml) behandelde twee-kamersystemen daarentegen kon deze gelocaliseerde IE180 expressie teruggevonden worden in alle geïnfecteerde neuronen op 24h na infectie. Dit toont aan dat IFN−α virusvermeerdering stillegt na de expressie van IE180 en voor de expressie van late virale eiwitten. De aanwezigheid van IE180 in alle geïnfecteerde neuronen wijst er ook op dat deze allemaal een functioneel viraal genoom ontvangen hebben. Een interessante bevinding was dat nog maar 45% van de geïnfecteerde neuronen deze IE180 expressie vertoonden op 5 dagen na infectie in de aanwezigheid van IFN−α. Deze vaststelling toont aan dat IFN−α de IE180 eiwitexpressie in de andere 55% van de geïnfecteerde neuronen kon onderdrukken tijdens deze 5 daagse incubatie periode. De afwezigheid van IE180 in deze 55% van geïnfecteerde neuronen impliceert bovendien dat deze neuronen geen detecteerbare virale eiwitexpressie meer vertoonden. In een volgend experiment werd onderzocht of de stabiel onderdrukte virale genomen konden gereactiveerd worden. We toonden aan dat toevoeging van forskolin, een product dat de cAMP niveau’s in de cel verhoogt en waarvan gekend is dat het reactivatie veroorzaakt in verschillende in vitro modellen, aan het medium na een 5 daagse incubatieperiode met IFN−α (500 U/ml) ervoor zorgde dat PRV reactiveerde in 50% van de neuronen die stabiel onderdrukte virale genomen bevatten en dit binnen een periode van 3 dagen, aangezien het spreiden van de infectie induceerde naar omgevende niet-neuronale cellen. Al deze data samen tonen aan dat de virale genomen die stabiel onderdrukt waren in de geïnfecteerde neuronen voldoen aan alle criteria die aanwezig zijn in de definitie van een latente infectie en bijgevolg kan geconcludeerd worden dat het toevoegen van IFN−α aan TG neuronen in vitro voldoende is om wild type PRV in een stabiele maar reactiveerbare latentie-achtige toestand te brengen in een meerderheid van geïnfecteerde neuronen. Dit is de eerste keer dat de latentie/reactivatiecyclus werd nagebootst in vitro op een fysiologisch relevante manier. Uit deze studie kunnen twee algemene besluiten getrokken worden over de interactie tussen PRV en TG neuronen in vitro: 1) Interactie van PRV-gD met zijn receptor op trigeminale neuronen van het varken induceert signalisatie via small Rho GTPasen en MAP kinasen. De activatie van signaaltransductiewegen leidt tot de vorming van ‘varicosities’. Deze viraal geïnduceerde ‘varicosities’ op een

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moment vroeg tijdens infectie (op het moment van aanhechting) kunnen vervolgens door het virus gebruikt worden als plaatsen om axonen te verlaten en op die manier het spreiden van het virus te vergemakkelijken. 2) Interferon alfa is een determinerende factor in de inductie van een reactiveerbare latente infectie van wild type PRV in een subset van trigeminale varkens neuronen in vitro. Het onderdrukt virale eiwitexpressie op een punt tussen immediate early en late virale eiwitexpressie op 24 h na infectie en het verwijdert alle detecteerbare virale eiwitexpressie in een meerderheid van geïnfecteerde neuronen op 120 h na infectie.

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Chapter 8: References

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Thompson, R. L., and N. M. Sawtell. 2001. Herpes simplex virus type 1 latency-associated transcript gene promotes neuronal survival. J Virol 75:6660-75. Thompson, R. L., and N. M. Sawtell. 2006. Evidence that the herpes simplex virus type 1 ICP0 protein does not initiate reactivation from latency in vivo. J Virol 80:10919-30. Tomishima, M. J., and L. W. Enquist. 2001. A conserved alpha-herpesvirus protein necessary for axonal localization of viral membrane proteins. J Cell Biol 154:741-52. Tomishima, M. J., and L. W. Enquist. 2002. In vivo egress of an alphaherpesvirus from axons. J Virol 76:8310-7. Tomishima, M. J., G. A. Smith, and L. W. Enquist. 2001. Sorting and transport of alpha herpesviruses in axons. Traffic 2:429-36. Tonomura, N., E. Ono, Y. Shimizu, and H. Kida. 1996. Negative regulation of immediateearly gene expression of pseudorabies virus by interferon-alpha. Vet Microbiol 53:271-81. Topp, K. S., L. B. Meade, and J. H. LaVail. 1994. Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus. J Neurosci 14:318-25. Torseth, J. W., and T. C. Merigan. 1986. Significance of local gamma interferon in recurrent herpes simplex infection. J Infect Dis 153:979-84. Townsend, J. J., and P. K. Collins. 1986. Peripheral nervous system demyelination with herpes simplex virus. J Neuropathol Exp Neurol 45:419-25. Trousdale, M. D., I. Steiner, J. G. Spivack, S. L. Deshmane, S. M. Brown, A. R. MacLean, J. H. Subak-Sharpe, and N. W. Fraser. 1991. In vivo and in vitro reactivation impairment of a herpes simplex virus type 1 latency-associated transcript variant in a rabbit eye model. J Virol 65:6989-93. Turner, A., B. Bruun, T. Minson, and H. Browne. 1998. Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J Virol 72:873-5. Udo, H., I. Jin, J. H. Kim, H. L. Li, T. Youn, R. D. Hawkins, E. R. Kandel, and C. H. Bailey. 2005. Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons. Neuron 45:887-901. Valitutti, S., S. Muller, M. Dessing, and A. Lanzavecchia. 1996. Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell receptor occupancy. J Exp Med 183:1917-21. van Leeuwen, H., M. Okuwaki, R. Hong, D. Chakravarti, K. Nagata, and P. O'Hare. 2003. Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT. J Gen Virol 84:2501-10.

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van Lint, A. L., L. Kleinert, S. R. Clarke, A. Stock, W. R. Heath, and F. R. Carbone. 2005. Latent infection with herpes simplex virus is associated with ongoing CD8+ T-cell stimulation by parenchymal cells within sensory ganglia. J Virol 79:14843-51. Verjans, G. M., R. Q. Hintzen, J. M. van Dun, A. Poot, J. C. Milikan, J. D. Laman, A. W. Langerak, P. R. Kinchington, and A. D. Osterhaus. 2007. Selective retention of herpes simplex virus-specific T cells in latently infected human trigeminal ganglia. Proc Natl Acad Sci U S A 104:3496-501. Vikman, K., B. Robertson, G. Grant, A. Liljeborg, and K. Kristensson. 1998. Interferongamma receptors are expressed at synapses in the rat superficial dorsal horn and lateral spinal nucleus. J Neurocytol 27:749-59. Villagra, N. T., J. Berciano, M. Altable, J. Navascues, I. Casafont, M. Lafarga, and M. T. Berciano. 2004. PML bodies in reactive sensory ganglion neurons of the Guillain-Barre syndrome. Neurobiol Dis 16:158-68. Vizi, E. S., J. P. Kiss, and B. Lendvai. 2004. Nonsynaptic communication in the central nervous system. Neurochem Int 45:443-51. Wakamatsu, K., H. Ogita, N. Okabe, K. Irie, M. Tanaka-Okamoto, H. Ishizaki, A. Ishida-Yamamoto, H. Iizuka, J. Miyoshi, and Y. Takai. 2007. Up-regulation of loricrin expression by cell adhesion molecule nectin-1 through Rap1-ERK signalling in keratinocytes. J Biol Chem 282:18173-81. Walters, J. N., G. L. Sexton, J. M. McCaffery, and P. Desai. 2003. Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells. J Virol 77:4043-59. Wang, K., T. Y. Lau, M. Morales, E. K. Mont, and S. E. Straus. 2005. Laser-capture microdissection: refining estimates of the quantity and distribution of latent herpes simplex virus 1 and varicella-zoster virus DNA in human trigeminal Ganglia at the single-cell level. J Virol 79:14079-87. Wang, Q. Y., C. Zhou, K. E. Johnson, R. C. Colgrove, D. M. Coen, and D. M. Knipe. 2005. Herpesviral latency-associated transcript gene promotes assembly of heterochromatin on viral lytic-gene promoters in latent infection. Proc Natl Acad Sci U S A 102:16055-9. Warner, M. S., R. J. Geraghty, W. M. Martinez, R. I. Montgomery, J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear. 1998. A cell surface protein with herpesvirus entry activity (HveB) confers susceptibility to infection by mutants of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus. Virology 246:17989. Whitley, R. J., and B. Roizman. 2001. Herpes simplex virus infections. Lancet 357:1513-8. Wigdahl, B., C. A. Smith, H. M. Traglia, and F. Rapp. 1984. Herpes simplex virus latency in isolated human neurons. Proc Natl Acad Sci U S A 81:6217-21.

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Zhou, G., E. Avitabile, G. Campadelli-Fiume, and B. Roizman. 2003. The domains of glycoprotein D required to block apoptosis induced by herpes simplex virus 1 are largely distinct from those involved in cell-cell fusion and binding to nectin1. J Virol 77:3759-67. Zhou, Z. H., D. H. Chen, J. Jakana, F. J. Rixon, and W. Chiu. 1999. Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions. J Virol 73:3210-8. Zhu, J., D. M. Koelle, J. Cao, J. Vazquez, M. L. Huang, F. Hladik, A. Wald, and L. Corey. 2007. Virus-specific CD8+ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation. J Exp Med 204:595-603. Zhu, P. C., A. Thureson-Klein, and R. L. Klein. 1986. Exocytosis from large dense cored vesicles outside the active synaptic zones of terminals within the trigeminal subnucleus caudalis: a possible mechanism for neuropeptide release. Neuroscience 19:43-54. Zwaagstra, J. C., H. Ghiasi, S. M. Slanina, A. B. Nesburn, S. C. Wheatley, K. Lillycrop, J. Wood, D. S. Latchman, K. Patel, and S. L. Wechsler. 1990. Activity of herpes simplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript. J Virol 64:5019-28

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CURRICULUM VITAE Personalia

Nick De Regge werd geboren op 7 maart 1980 te Deinze. In 1998 beëindigde hij zijn secundaire studies (richting Wetenschappen-Wiskunde) aan het Don Bosco Instituut te Zwijnaarde. In datzelfde jaar startte hij zijn universitaire studies aan de toenmalige Faculteit Landbouwkundige

en

Toegepaste

Biologische

Wetenschappen

(nu

Bio-

ingenieurswetenschappen) aan de Universiteit Gent en in 2003 behaalde hij het diploma van Bio-ingenieur in de Cel- en Genbiotechnologie met grote onderscheiding. Vanaf september 2003 tot december 2004 beschikte hij over een doctoraatsbeurs van het Bijzonder Onderzoeksfonds van

de Universiteit

Gent

die kaderde in een

Geconcerteerde

Onderzoeksactie (GOA) aan het Laboratorium voor Virologie, Vakgroep Virologie, Parasitologie en Immunologie aan de Faculteit Diergeneeskunde, Universiteit Gent. Sinds januari 2005 tot op heden is hij houder van een specialisatiebeurs van het Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (IWTVlaanderen). Dit onderzoek handelde over de in vitro interacties tussen het pseudorabies virus en neuronen van het trigeminaal ganglion waarbij de focus lag op het neuronaal spreiden en het instellen van latentie.

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Publicaties Publicaties in internationale wetenschappelijke tijdschriften De Regge, N., H. W. Favoreel, K. Geenen, and H. J. Nauwynck. 2006. A homologous in vitro model to study interactions between alphaherpesviruses and trigeminal ganglion neurons. Veterinary Microbiology 113:251-255. De Regge, N., H. J. Nauwynck, K. Geenen, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, T. C. Mettenleiter, and H. W. Favoreel. 2006. Alpha-herpesvirus glycoprotein D interaction with sensory neurons triggers formation of varicosities that serve as virus exit sites. Journal of Cell Biology 174:267-275. Geenen, K., H. J. Nauwynck, N. De Regge, K. Braeckmans, and H. W. Favoreel. 2007. Brn3a suppresses pseudorabies virus-induced cell death in sensory neurons. Journal of General Virology 88:743-7. In preparation: De Regge, N., H.J. Nauwynck, and H.W. Favoreel. Interferon alpha induces a latent alphaherpesvirus infection in trigeminal ganglion neurons in vitro. De Regge, N., J. Ficinska, P. Spear, H. Nauwynck, K. Bienkowska-Szewczyk, H.W. Favoreel. Transfection of pseudorabies virus glycoprotein D in nectin-1 expressing CHO cells results in Rho GTPase-dependent formation of filopodia-like structures.

Octrooi aanvragen EPO 07011225: Viral Latency Model, gefiled op 8 juni 2007 Inventors: Nick De Regge, Herman Favoreel, Hans Nauwynck

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Abstracts De Regge, N., H.W. Favoreel, K. Geenen, and H.J. Nauwynck. 2005. A homologous in vitro model to study interactions between alphaherpesviruses and trigeminal ganglion neurons. 2nd ESSV Veterinary Herpesvirus Symposium, Gent, Belgium. De Regge, N., H.W. Favoreel, K. Geenen, and H.J. Nauwynck. 2005. Pseudorabies virus infection of porcine trigeminal ganglion neurons induces the formation of synaptic boutons along the axon that may constitute sites of virus release. 30th International Herpesvirus Workshop, Turku, Finland. De Regge, N., H.J. Nauwynck, K. Geenen, and H.W. Favoreel. 2005. Pseudorabies virus infection induces the formation of pre-synaptic boutons along axons of porcine trigeminal ganglion neurons that may serve as virus release sites. BSCDB meeting (Belgische vereniging voor cel- en ontwikkelingsbiologie): Neuroglia, 15 oktober, Diepenbeek, Belgium. De Regge, N., H.J. Nauwynck, K. Geenen, and H.W. Favoreel. 2005. Infection of trigeminal ganglion neurons with the alphaherpesvirus pseudorabies virus induces the formation of synaptic boutons that may serve as axonal exit sites for the virus. Symposium of the Belgian Society for Microbiology, 18 november, Brussel, Belgium. Geenen, K., H.J. Nauwynck, N. De Regge, and H.W. Favoreel. 2006. Expression of the cellular anti-apoptotic factor Brn-3a correlates with increased resistance of trigeminal ganglion neurons towards pseudorabies virus-induced cell death. 31st International Herpesvirus Workshop, Seattle, Washington, VS. De Regge, N., H.J. Nauwynck, K. Geenen, C. Krummenacher, G.H. Cohen, R.J. Eisenberg,T.C. Mettenleiter, and H.W. Favoreel. 2006. Interaction of gD of PRV with axons of trigeminal ganglion neurons triggers the formation of synaptic varicosities that serve as virus exit sites. 31st International Herpesvirus Workshop, Seattle, Washington, VS.

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De Regge, N., H.J. Nauwynck, K. Geenen, C. Krummenacher, G.H. Cohen, R.J. Eisenberg,T.C. Mettenleiter, and H.W. Favoreel. 2006. Interaction between the gD envelope protein of pseudorabies virus and trigeminal ganglion neurons triggers the formation of synapses along the neuronal axons that may serve as virus exit sites. Symposium of the Belgian Society for Microbiology, 24 november, Brussel, Belgium. De Regge, N., J. Ficinska, P.G. Spear, H.J. Nauwynck, K. Bienkowska-Szewczyk, and H.W. Favoreel. 2007. Transfection of pseudorabies virus glycoprotein D in nectin-1 expressing CHO cells results in small Rho GTPase- and MAP kinase-dependent formation of filopodialike structures. 32th International Herpesvirus Workshop, Asheville, North Carolina, VS

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DANKWOORD De voorbije vier jaar aan het Laboratorium voor Virologie zijn voorbij gevlogen. Dat kwam deels door het interessante onderwerp van mijn onderzoek, maar nog meer door de vele mensen die ik hier ontmoet heb en die gedurende deze periode in meer of mindere mate hebben bijgedragen tot het tot stand komen van deze thesis. Het is dan ook met plezier dat ik hen op het einde van deze periode wil bedanken. Eerst en vooral zou ik mijn beide promotoren, Prof. Dr. Herman Favoreel en Prof. Dr. Hans Nauwynck, willen bedanken. Herman, dit alles is een goeie vier jaar geleden begonnen met een telefoontje van jou, terwijl ik op het labo van mijn thesis beestjes aan het instuderen was voor het examen gewasbescherming, om te vragen of ik niet geïnteresseerd was om eventueel een doctoraat te doen op het Labo voor Virologie. Reeds vanaf dat eerste gesprek had ik er een goed gevoel bij en dat heeft me niet bedrogen. Herman, jij hebt mij op een onnavolgbare wijze door m’n doctoraat geloodst. Je stond steeds klaar om al m’n vragen te beantwoorden met je enorme literatuurkennis, je kwam steeds op de proppen met nieuwe ideeën om moeilijk lopende experimenten aan te pakken en de efficiëntie waarmee jij teksten leest en corrigeert is ongeëvenaard. Je hebt me heel veel bijgebracht over het belang van een continue literatuurstudie, het opzetten van goeie experimenten en het schrijven van teksten! Ik wil je ook bedanken voor de vrijheid die je me steeds gelaten hebt om de dingen op mijn manier aan te pakken en voor je steun bij alles wat ik ondernomen heb. Je hebt er altijd voor gezorgd dat ik over alle producten kon beschikken die ik nodig had (dacht nodig te hebben), iets wat waarschijnlijk niet altijd evident was. Ik heb ook altijd geapprecieerd dat je steeds met een open geest naar me geluisterd hebt, over alles konden we discussiëren en samen zijn we steeds tot de beste oplossing gekomen. Ook bedankt voor de subtiele manier waarop je me af en toe wist af te remmen of in te tomen, je hebt me op die manier zeker een aantal flaters bespaard. Daarnaast wil ik je ook bedanken omdat je er steeds voor geijverd hebt dat ik meekon naar het jaarlijks herpesviruscongres. Voor mij zijn dit drie onvergetelijke uitstappen geweest die m’n motivatie voor het onderzoek alleen maar hebben aangewakkerd en waardoor ik toch ook een (mooi) stukje van de wereld heb gezien. Naast al dit wetenschappelijke wil ik je zeker ook bedanken voor de vele leuke babbels, voor de motiverende gesprekken over de toekomst en voor je goede raad. Ik wens je heel veel succes

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met de verdere uitbouw van je onderzoeksgroep en hoop dat we ook in de toekomst nog kunnen samenwerken. Hans, jou wil ik bedanken voor de interesse in mijn werk met de neuronen en voor de boeiende discussies tijdens de seminars. Bedankt dat je er altijd voor gezorgd hebt dat de aankoop van varkens en producten geen hinderpaal was tijdens mijn onderzoek. Ik bewonder de manier waarop jij een labo van die grootte draaiende houdt en zoveel mensen kunt begeleiden. Het kan niet anders dan dat je dag en nacht met het labo bezig bent. Respect. Die drive die van jou uitgaat, of laten we het power noemen, heeft me altijd gemotiveerd. Daarnaast wil ik je nog bedanken voor een aantal legendarische momenten tijdens de herpesviruscongressen. Je hebt er altijd voor gezorgd dat er tijdens deze congressen, naast het wetenschappelijke, ook plaats was voor een gezellige uitstap als collega's onder elkaar. Prof. Dr. Kristien van Reeth wens ik te bedanken voor haar interesse in mijn onderzoek met neuronen en voor haar kritische opmerkingen tijdens de seminars. Verder wil ik ook de leden van mijn begeleidingscommissie (Prof. Dr. T. Mettenleiter, Prof. Dr. J. Piette, Dr. P. Vanhoenacker en Prof. Dr. E. Meyer) bedanken voor de tijd die ze hebben gestopt in het doornemen, bijsturen en evalueren van dit proefschrift. Graag wil ik ook onze technology-developer Dr. Sven Arnouts en Dr. Dominic De Groote en Dr. Lieve Nuytinck van de afdeling Technologie Transfer bedanken voor het geloof in de commerciële waarde van dit onderzoek en de hulp bij het uitwerken van de octrooiaanvraag. Dr. Kevin Braekmans van de faculteit Farmaceutische Wetenschappen (Universiteit Gent) bedank ik voor de hulp bij het gebruik van de UV-laser van hun confocale microscoop. De Universiteit Gent wil ik bedanken voor de financiering tijdens het begin van mijn doctoraat via een beurs van het Bijzonder Onderzoeks Fonds. Het IWT-Vlaanderen bedank ik voor de specialisatie-beurs die ze mij toegekend hebben. Deze heeft me toegelaten om sinds 2005 van mijn passie mijn beroep te maken. Een heel belangrijk woord van dank wil ik richten aan het technisch personeel van het labo virologie. Zij hebben een groot deel van het praktisch werk, zoals het splitsen van cellen, het opgroeien van virusstocks, de sterilisatie van het materiaal,... op hun genomen waardoor ik me ten volle kon concentreren op m’n werk met de neuronen. Met wie kan ik anders beginnen dan met Carine... Carine, al heel snel nadat ik op het labo gearriveerd was, had ik in de gaten dat zo’n beetje plat Eeks en een vlot mondje Zwijnaards wel goed bij elkaar pasten. Ik had dan ook snel m’n vaste plaats aan de flow gevonden en zo hebben we de voorbije 4 jaar ontelbare uren in elkaars gezelschap doorgebracht. Ik wil je bedanken voor de grote

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interesse in m’n werk, je gedrevenheid om altijd mee te denken als ietskes was mislukt en je hulp met kleuringen, titraties, SN testen en ga zo maar door. Altijd was je bereid om me te helpen, ook al stond je agenda reeds volboekt. Maar daarnaast wil ik je evenzeer bedanken voor de vele fijne babbels, de muzikale omlijsting en zo nu en dan wat medium in m’n oog :-). Jij hebt ervoor gezorgd dat ik het praktisch werk altijd met het grootste plezier heb gedaan. Lieve, jou wil ik graag bedanken voor het splitsen van vele ST’s en het klaarmaken van verschillende voedingsmedia, maar evenzeer voor je aangename gezelschap wanneer we met heel de bende op stap gingen. Chantal, jou wil ik bedanken voor de verschillende virusstocks die je voor mij hebt opgegroeid en getitreerd, voor het planten van allerlei cellen en het uitvoeren van verschillende SN testen. Nele, bedankt voor de toffe gesprekjes en voor het klaarleggen van de dekglaasjes voor m’n neuronenculturen. Chris en Melanie, jullie waren op het eerste zicht niet zo nauw betrokken bij mijn onderzoek, maar toch bedankt voor het praktische werk dat mij het leven veel eenvoudiger heeft gemaakt. Natasha, voor jou moet het nog allemaal beginnen maar ik sta alvast versteld van de vele technieken die je op zo’n korte termijn al onder de knie hebt gekregen en van de zelfstandigheid waarmee je de zaken aanpakt. En dan zijn er natuurlijk nog twee, twee die ik speciaal tot het laatste heb gehouden. Fernand en Geert O., merci voor het vele werk dat jullie voor mij hebben verricht: het steriliseren van al dat materiaal, het halen van talloze biggen, den autoclaaf eens extra in gang steken als ik eens iets was vergeten, de hulp bij het bricoleren met van alles en nog wat, we waren 3 goeie doe-het-zelvers samen. Ook bedankt voor de vele toffe gesprekken (een bezoekje aan de keuken bracht altijd opluchting wanneer het eens allemaal te veel werd), zo nu en dan een fratske, voor het in de gaten houden van ‘de nieuwe lichting’ en het waakzame oog als ik onder de middag buiten m’n boterhammekes ging opeten. Mieke wil ik bedanken voor de vele administratieve zaken die ze altijd met het grootste enthousiasme voor mij heeft geregeld. Gert, jou wil ik bedanken voor de vele bestellingen die je voor mij gedaan hebt en dan vooral voor degene buiten de vaste besteldagen. Ze waren allemaal héél dringend en belangrijk :-), alsof je nog geen ander werk genoeg had. Bedankt voor de toffe momenten, een bezoekje aan het secretariaat was dankzij jullie altijd een moment van verademing. Dirk, ik wil je bedanken om zo nu en dan eens een nieuwe virus-scanner of een printer op m’n computer te komen installeren. Ook heel erg bedankt voor de hulp bij de lay-out van m’n doctoraat en voor het last minute regelen van de drukker. Dat heeft me veel extra stress bespaard!

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Marijke, voor jou moet het heel lastig geweest zijn om op mijn vraag m’n bureau nooit af te kuisen. Hopelijk heb je geen slapeloze nachten overgehouden aan dat stort :-). Ik wil je ook heel erg bedanken voor die vriendelijke en opgewekte ‘goeiemorgen’ telkens ik toekwam, het herinnerde me er altijd aan dat m’n ochtendhumeur al lang genoeg had geduurd. Natuurlijk wil ik ook de vele collega doctoraatstudenten bedanken. Gedurende de voorbije 4 jaar heb ik een aantal mensen zien vertrekken, maar vooral heb ik er een hele groep jonge mensen zien bijkomen. Naast het feit dat de continue uitbreiding ervoor gezorgd heeft dat het steeds moeilijker werd om een plekje aan de flow te vinden, een plaatsje achter de fluorescentiemicroscoop te versieren of je cellen kwijt te kunnen in de broedstoof, heeft het er vooral voor gezorgd dat er op de ‘virologie’ altijd (en dat mag je zo ongeveer letterlijk nemen) een plezante, joviale en motiverende sfeer heerste. Laat mij beginnen met de oudgedienden. Kristin, jou wil ik bedanken omdat je me 4 jaar geleden begeleid hebt tijdens de eerste stappen van m’n doctoraatsonderzoek. Jij het me vakkundig geleerd hoe ik de TGs uit varkens moest isoleren en hoe de neuronen in cultuur moesten worden gebracht. Op die moment leek dat evident, maar na vele uren literatuurstudie en het bijwonen van verschillende congressen besef ik dat dit niet het geval is en prijs ik me gelukkig dat jij dat reeds allemaal op punt had gesteld. Geert V.M. en Peter D., jullie continue interesse en gemotiveerde manier van werken heeft ervoor gezorgd dat ik snel op het juiste onderzoeksspoor zat. Van jullie heb ik ook geleerd dat er naast het ‘werk’ nog tijd moet zijn voor andere zaken zoals vissen, voetbal en cyclocross bijvoorbeeld :-), en dat op tijd en stond een goeie pint pakken met de collega’s belangrijk is voor de goeie sfeer. Pas na de verschillende whisky’s aan den toog van ‘De pub’ op het einde van de eerste labo-uitstap ben ik me echt op het labo gaan thuisvoelen. Merci daarvoor! Peter M. en Steven, jullie waren de eersten die me geholpen hebben bij het euthanaseren van de biggen. Bedankt hiervoor, maar evenzeer voor de plezante babbels. Van Peter heb ik geleerd dat nog niemand gestorven is van een nachtje doorwerken, iets wat je zou kunnen betwijfelen als ik aan een aantal nachtelijke passages van Steven op het labo terugdenk :-). Ook Geoffrey wil ik bedanken voor zijn grote interesse in mijn werk, maar ook voor zijn gedrevenheid om aan te tonen dat R.S.C.A. niet de best club van ’t land is. Karen, bedankt voor je doorzettingsvermogen om me het belang van de bioveiligheid bij te brengen. Het heeft z’n vruchten afgeworpen, meestal toch :-). En dan zijn er natuurlijke de vele mensen die samen met mij zijn begonnen of erna de groep hebben vervoegd. Het is onbegonnen werk om iedereen persoonlijk te bedanken, neem het me dan ook niet kwalijk als ik iemand vergeet of niet uitvoerig genoeg bedank. Sarah G., eerst en vooral bedankt om me te laten helpen bij het inspuiten van je varkens, hoe zou ik me anders ooit een ‘echte :-)’doctor in de

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diergeneeskundige wetenschappen kunnen voelen. Maar vooral bedankt voor de vele middagpauzes waarin je me gezelschap hebt gehouden, voor iemand die zo veel boterhammekes eet als ik is dat niet te onderschatten. Bedankt voor de vele fijne babbels, je luisterend oor waarbij ik altijd met m’n frustraties terecht kon en je steun in moeilijker momenten. Ik wens je bijzonder veel succes bij alles wat je onderneemt en bij het afwerken van je mooi, maar moeilijk en arbeidsintensief doctoraatsonderzoek. Sarah C., ook jou wil ik bedanken voor de de toffe gesprekken, de organisatie van verschillende avondjes uit, de nicknick’s als ik door de gang liep, de veel te straffe cocktails net voor IWTproefverdedigingen,... Ik denk niet dat ik hier één avond of weekend ben komen werken zonder dat jij hier ook was. Jouw inzet en motivatie zijn een voorbeeld voor velen, ik hoop dat je hiervoor beloond wordt met een mooi doctoraat. Hannah en Ann D., het was plezant om samen met twee bekende gezichten aan die toen nieuwe uitdaging te kunnen beginnen. Bedankt voor de interesse en de plezante babbels. Veel succes met het beëindigen van jullie werk. David en Filip, merci dat jullie altijd klaarstonden om te helpen bij het euthanaseren van mijn varkens en voor de diervriendelijke manier waarop jullie dit altijd hebben gedaan. David, ook merci voor de toffe babbels, een eens iets vettiger opmerking op zijn tijd zorgde altijd voor de nodige ambiance. Filip, chance dat gij er waart om die hoop enthousiaste dames in bedwang te houden, zeker Sarah C. :-). Ook bedankt voor het uitvoeren van de IFN-α testen. Kristien van den immuno, ik weet niet meer precies hoe ik jou heb leren kennen maar waarschijnlijk zal je wel eens iets nodig gehad hebben van op de viro. Ik wil je hartelijk bedanken voor de interesse in mijn werk, de vele fijne gesprekken en je subtiele lach. Je hebt meermaals m’n dag opgevrolijkt. Kom maar af als je nog eens iets nodig hebt, maar ik heb het gevoel dat het misschien eerder omgekeerd zal zijn :-). Matthias D., Céline en Hanne, met jullie komst kwamen er op slag drie ‘oude’ bekenden bij. Matthias, ik heb je altijd geapprecieerd voor je oprechte interesse in mijn onderzoek en voor je doorzettingsvermogen, je bent niet voor niets de Harry Blotter van het labo :-). Ook merci voor de vele toffe gesprekken over het vissen en de voetbal. Bijt nog even door met je onderzoek, je staat er beter voor dan je zelf denkt! Céline en Hanne, bedankt voor de interesse naar de toestand van mijn neuroontjes, voor de vele open en geanimeerde gesprekken en de motiverende woorden. Dankzij jullie is de moleculaire kennis op het labo fameus toegenomen, bedankt voor de vele goeie tips! Céline, als je nog eens met de go-kart wil crossen of gaan raften, je weet mij te vinden. Tis misschien niet altijd even verantwoord, maar leute gegarandeerd. Dan is er natuurlijk nog ons vedet. Wanderke, ik ben er nog altijd niet uit hoe ze zoveel energie in zo’n

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klein bazeke hebben gekregen. Een Wanderke in form is onbetaalbaar voor de sfeer. We hebben samen binnen en buiten het labo vele toffe momenten beleefd. Bedankt (voor die bloeeeemen)! We gaan zeker samen nog eens een stapke in de wereld zetten. Blijf gaan voor je onderzoek en vindt je eigen weg, zoveel inzet en motivatie wordt uiteindelijk altijd beloond! Merijn en Iris, jullie waren toffe bureau-genoten tijdens de laatste jaren. Merijn, ik vind het chique dat je de ‘guts’ hebt om op zo een zelfstandige manier een nieuwe wending aan je onderzoek te geven. Veel succes! Ook mijn voormalige thesisstudente Kristen wil ik graag bedanken. Dankzij haar heb ik geleerd dat mensen begeleiden veel tijd, inzet en betrokkenheid vraagt en dat heeft m’n respect voor mijn begeleiders nog doen toenemen. Veel succes met je doctoraatsonderzoek over HIV. Evelien, Els, Annebel, Annelies, Annick en Mathias C., bedankt voor de fijne momenten op of buiten het labo. Gerald, Meezanur, Kalina and Constantinos, I wish you guys a lot of success during the final stages of your PhD studies. Gerald, I admire your way of working and the efficiency with which you produced those interesting publications. It was a pleasure to have you as a companion in the meeting room while we were writing our thesis. You are a great scientist, the best of luck to you when you go back to Afrika. Meezanur, I will never forget you asking: ‘How is life, Nick?’ during my lunch break :-). I hope everything turns out well for you. Kalina, I want to thank you for your friendliness and great enthousiasm. Hopefully my neurons can come in handy for you. En dan is er natuurlijk nog de nieuwe garde. An S., Marc, Sjouke, Debbie, Miet, Joao en Uladzimir, jullie staan nog helemaal aan het begin van jullie onderzoek. Ik wil jullie alle succes wensen, ga ervoor, laat jullie niet ontmoedigen door de tegenslagen die er zeker zullen komen en vergeet vooral niet om veel plezier uit jullie werk te halen! Nina, jij bent de laatste in dit rijtje. Lang heb ik mijn best gedaan om het werk met de neuronen voor mezelf te houden. Nu ben ik blij dat jij er bent om het verder te zetten. Een welgemikte godverd**** wanneer er een kleinigheid fout liep en je goeie eerste neuronenculturen hebben me er al van overtuigd dat jij de geschikte persoon bent voor dit werk. Je kan je verwachten aan een hele hoop frustratie maar hou in je achterhoofd dat je dankzij die cellen interessante en relevante resultaten bekomt. Heel veel succes en weet dat ik altijd bereid ben om je bij te staan met raad en daad. Dan wil ik graag ook al mijn vrienden en vriendinnen van buiten het labo bedanken omdat zij steeds de moeite gedaan hebben om interesse te tonen voor mijn onderzoek, maar vooral voor de vele gezellige etentjes, de bemoedigende gesprekken, de pinten en cocktails aan den toog, een avondje snooker of pool, een fitness of pretpark bezoekje, een ritje met de moto,... Bedankt voor de vriendschap en de ontspannende momenten.

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Ten slotte wil ik zeker ook mijn familie bedanken voor de steun en interesse naar wat ik daar nu toch allemaal aan het uitsteken was op de veeartsenijschool. Merci voor de vele toffe familiefeesten, de ‘grote klakken’, de etentjes en uitstapjes met neven en nichten en het feit dat ik altijd op jullie kon rekenen. Willy, merci voor de magnifieke kasters, zonder die had ik nooit m’n belangrijkste prestatie van het jaar kunnen leveren, kampioen van ‘den ouden eind’, tis nie niets é :-). Graag wil ik ook nog twee personen bedanken die er op de dag van m’n verdediging niet bij zijn, maar die me altijd hard gesteund hebben en van wie ik weet dat ze bijzonder fier zouden geweest zijn. Mé en Pé, bedankt! Ik ben bijna aan het einde van m’n dankwoord gekomen en dan wordt het tijd om de belangrijkste mensen aan bod te laten komen. Dat zijn meestal degene van wie het zo vanzelfsprekend lijkt dat ze er altijd voor je zijn dat je vergeet om die daar op tijd en stond voor te bedanken. Ma en pa, jullie continue steun en interesse in alles wat ik doe betekent veel voor mij. Altijd staan jullie voor mij klaar, niets was ooit te veel gevraagd, één telefoontje is altijd voldoende. Bedankt voor alle kansen die jullie mij geboden hebben! Ik besef maar al te goed dat ik met m’n gat in de boter ben gevallen. Broerke, merci voor de hulp bij het maken van een aantal figuren die in deze thesis staan, voor de plezante pool-avonden waarbij ik je altijd fameus op je doos kon geven en je steun in moeilijke tijden. Vooraleer dit dankwoord af te sluiten moet ik nog één persoon bedanken, de allerbelangrijkste van allemaal. Ilse, het valt moeilijk te beschrijven wat jouw inbreng geweest is in dit werk. Bepaalde stukken hebben bloed, zweet en tranen gekost, maar het was jij die me altijd gemotiveerd hebt om door te bijten en er alles uit te halen, ook al wou dat zeggen dat jij tijdens die periodes alle taken op je schouders moest nemen en ik er vaak niet was. Jouw grote steun en rotsvast geloof in mijn kunnen waren een belangrijke bron van motivatie, tis plezant om te weten dat er iemand is die 100% achter je staat. Daarnaast slaagde je er ook wonderwel in om alles te relativeren en me niet te laten vergeten dat er nog belangrijker dingen zijn in het leven. Bedankt voor de fijne tijd, nu toch al meer dan 9 jaar, en ik kijk vol verwachting uit naar wat de toekomst ons nog brengen zal. Ondanks alles ben je altijd je lieve en opgewekte zelf gebleven die altijd klaar staat om alles voor een ander te doen. Prutske, ge zijt een straf madam, mijn madam! Nick