R e vie w

Sabrina Schreiner*, Peter Wimmer & Thomas Dobner Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Martinistrasse 52, 20251 Hamburg, Germany *Author for correspondence: Tel.: +49 40 48051 309 n Fax: +49 40 48051 302 n sabrina.schreiner@hpi. uni-hamburg.de

Adenoviruses

up to 1 × 104 viral particles are released upon host cell lysis [7] . Protein degradation by cellular E3 ubiquitin ligases

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The family of Adenoviridae consists of approximately 100 different so far known serological types, which divide into five genera depending on the host range (Box 1) [1–3] . To date, 54 human adenoviruses (Ad) have been identified and further subgrouped into seven species, A–G, according to sequence homology, hemagglutination and oncogenicity in immunosuppressed rodents [3,4–6] . Ad are nonenveloped viruses with an icosahedral capsid of 80–110 nm containing a linear dsDNA [7] . Depending on the serotype, the viral genome includes transcription units encoding approximately 40 regulatory and structural proteins and usually two ncRNAs (virusassociated RNAs [VA-RNAs]). Together, these include five early (E1A, E1B, E2, E3 and E4), two delayed (IX and IVa2) and one major late transcription unit (MLTU) [7] . Ad can infect a wide range of cell types, including resting cells, differentiated epithelial cells and cells derived from the nervous system [7,8] . Upon receptor-mediated viral uptake, the genome is imported into the nucleus prior to early E1A gene expression [9–12] . E1A expression leads to the transcription of E1B to E4 gene products, which trigger cell cycle progression and simultaneously block apoptosis and growth arrest [7,13,14] . During the late phases of Ad infection, the host cell mRNA transport and translation pathways are shut down, while concurrently viral late mRNAs are efficiently synthesized, transported to the cytoplasm and preferentially translated [6,15–17] . Completing the viral lifecycle, viral DNA is packaged in the nucleus and

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Eukaryotic cells orchestrate constant synthesis and degradation of intracellular components, including soluble proteins and organelles. The two major intracellular degradation pathways are the ubiquitin/proteasome system and autophagy. Whereas ubiquitin/proteasome system is involved in rapid degradation of proteins, autophagy selectively removes protein aggregates and damaged organelles. Failure of these highly adjusted proteolytic systems to maintain basal turnover leads to altered cellular homeostasis. During evolution, certain viruses have developed mechanisms to exploit their functions to facilitate their own replication, prevent viral clearance and promote the outcome of infection. In this article, we summarize the current opinion on adenoviruses (Ad) and molecular host cell targets, extending on recent evidences for protein degradation pathways in infected cells. We describe recently identified connections between Ad-mediated proteolysis and viral replication with main emphasis on the function of certain Ad proteins.

Review

Future Microbiology

Adenovirus degradation of cellular proteins

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In general, ubiquitin ligases are known to maintain cellular homeostasis on a posttranslational level by the covalent conjugation of ubiquitin (Ub), a small 8 kDa protein, onto target proteins. These biological processes are orchestrated by activating (E1), conjugating (E2) and ligating (E3) enzymes [18] . E1 provides a thioester bond between its conserved cysteine residue and the C-terminal carboxyl group of Ub in an ATPdependent manner. The activated molecule is passed by transthiolation reaction to a reactive cysteine residue of the specific E2 conjugating enzyme, recruited by E1. In cooperation with an E3 ligation enzyme, E2 conjugates Ubs to either the N-terminus or internal lysine residues of targeted proteins [19] . E3 ubiquitin ligases have been classified into three groups: the HECT domain type, the single subunit RING finger type, and the multisubunit RING finger type. HECT ligases transfer activated Ub to the E3 cysteine residue before ligation onto targeted proteins, whereas RING ligases do not posses a catalytic function and act as scaffolds for the conjugation reaction by bridging the E2/Ub complex and its substrate [20] . The best characterized mammalian Cullindependent ligase is the SCF ligase complex with an adaptable mechanism for discriminating between substrates (Figure 1A) [21,22] . The core complex is composed of a Cullin1 subunit, that

10.2217/FMB.11.153 © 2012 Future Medicine Ltd

Future Microbiol. (2012) 7(2), 1–xxx

Keywords adenovirus n autophagy Daxx n degradation n DNA ligase IV n E1B-55K n E3 ubiquitin ligase n integrin a3 n proteasome n E4orf6 n p53 n Mre11 n TopBP1 n

n BLM n

part of

ISSN 1746-0913

1

Review

Schreiner, Wimmer & Dobner

Box 1. Adenoviruses. The Adenoviridae family consists of approximately 100 different serotypes, which are divided into five genera depending on the host range (Mastadenoviridae, Aviadenoviridae, Atadenoviridae, Siadenoviridae and Ichtadenoviridae) Fifty four human Adenoviruses (HAd) are subdivided into species A–G Adenoviruses are nonenveloped viruses with an icosahedral capsid of 80–110 nm Adenoviral genes are encoded in a dsDNA of 20–40 kbp in length The Adenovirus genome includes transcription units encoding for approximately 40 regulatory and structural proteins and two ncRNAs (virus-associated RNAs) Adenovirus transcription units encode for five early (E1A, E1B, E2, E3 and E4), two delayed (IX and IVa2) and one major late transcription unit (MLTU) Adenoviruses are known to infect a wide range of cell types, mainly postmitotic resting cells, differentiated epithelial cells and most likely cells derived from the nervous system Adenovirus DNA packaging takes place in the nucleus, viral lifecycle is completed and up to 1 × 10 4 viral particles are released upon host cell lysis

complex (Figure 1A) [27–29] . In line with this, the founding members of the Cullin5 family of E3 ubiquitin ligases are the SOCS proteins [30] . Sequence comparison of the VHL and SOCSbox proteins revealed that they each include a sequence motif with a conserved BC-box motif, which is required for binding to the Elongin BC complex (Figure 1) . To date, more than 30 different BC-box proteins have been identified and a number of these have been shown to function as components of Cullin2 and Cullin5 ubiquitin ligases [26] .

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functions as a molecular scaffold that simultaneously interacts with the crucial adaptor subunit Skp1, with a RING finger protein (Rbx1, also known as Roc1 or Roc2) and a specific E2 enzyme or Ub conjugating enzyme (Ubc), such as Ubc3, Ubc4 or Ubc5 [20] . Skp1 binds to one of many F-Box proteins, and each of those F-Box proteins appears to be matched with a discrete number of specific substrates through a protein–protein interaction domain [23,24] . The most notable F-box protein is Skp2 with a capacious substrate repertoire including cell cycle and apoptosis regulators. These factors are bound either directly or via adaptor proteins and presented to the ubiquitination unit of the SCF complex [25] . Once all of the F-box proteins are reported, we will presumably form an adequate impression of the dimension of strategies adjusting substrate specificity and modulatory flexibility encoded in the regulatory components of ubiquitin ligases [19] . Recent evidence highlighted that members of the Cullin2 and Cullin5 containing E3 ubiquitin ligases are involved in regulating diverse cellular processes [23] . Cullin2 and Cullin5 ubiquitin ligases include the heterodimeric Elongin BC complex, composed of the Elongin C protein, which is similar in sequence to the Skp1 ubiquitin ligase subunit, and Elongin B protein (Figure 1A) . In the context of Cullin2 and Cullin5 ubiquitin ligases, the Elongin BC complex functions as an adaptor that links a BC box substrate recognition subunit to either a Cullin2/Rbx1 or a Cullin5/Rbx2 complex that uses the RING finger domains of the related Rbx1/Rbx2 proteins to recruit and activate an E2 ubiquitinconjugating enzyme for ubiquitination of substrates [26] . One of the first identified members of the Cullin2 family of E3 ubiquitin ligases was the von Hippel-Lindau (VHL) tumor suppressor

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Future Microbiol. (2012) 7(2)

Adenovirus-dependent E3 ubiquitin ligases

Viruses have acquired ways to optimize their replication by reprogramming regulatory pathways in infected cells [31] . For example, Ads have gained functions that modulate doublestrand break repair, apoptosis, cellular gene expression and host cell immune responses, all helping to promote efficient virus replication (Figure 2) . The first transcripts expressed by Ad are E1A and E1B. E1A is required for driving the host cell into S-phase during the early steps of infection, and for transcriptional activation of other Ad early genes [32–34] . Subsequently, E1B-55K supports efficient viral replication by inhibiting antiproliferative processes induced by the host cell and by stimulating the cytoplasmic accumulation and translation of viral late mRNAs [16,17,35] . However, the multiple functions of E1B55K require its interaction with E4orf6. So far, several reports have demonstrated that Ad5 E4orf6 connects Ad5 E1B-55K to an E3 ubiquitin ligase complex in the nucleus, containing cellular factors Rbx1/Roc1/Hrt1, Cullin2/5, Elongin B and C (Figur e 1 & B ox 2) [36–38] . It appears that in this E3 ligase complex, the viral component E1B-55K serves as future science group


p27

Review

HIF

Cks 1

Ubiquitin

Skp 2 Skp 1

Elongin B/C

Rbx1

Cullin 1

Ubiquitin

VHL

Rbx1

Cullin 2

SCF-dependent E3 ubiquitin ligase complex

VHL-dependent E3 ubiquitin ligase complex

Substrate

E4orf6 Elongin B/C

Rbx1

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Cullin 2/5

Ubiquitin

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E1B-55K

E1B-55K/E4orf6-dependent E3 ubiquitin ligase complex

TopBP1

Ubiquitin

Ad5 E1B-55K

rP

Daxx

Ad12 E4orf6

Elongin B/C

Elongin B/C Cullin 5

Rbx1

Rbx1

E4orf6-dependent E3 ubiquitin ligase complex

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E1B-55K-dependent E3 ubiquitin ligase complex

Cullin 2

Ubiquitin

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Figure 1. Overview of SCF, VHL and adenovirus-mediated ubiquitin ligase complexes. (A) Parallels between SCF and VHL ubiquitin ligase complexes modified from Querido et al. [38] . (B) E1B-55K/E4orf6 E3 ubiquitin ligase and so far proposed variations of either (Ad5) E4orf6- or (Ad12) E1B-55K-independent ubiquitin ligase complexes. E1B-55K: Early region 1 55K protein; E4orf6: Early region 4 open reading frame 6 protein; p27: p27Kip 1. Ad: Adenovirus; SCF: Skp1-Cullin1/Cdc53-F-box; VHL: von Hippel Lindau.

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the substrate recognition unit while the other viral protein E4orf6 assembles the cellular components. This high molecular mass complex (800–900 kDa) sequesters specific cellular substrates into the ubiquitin/proteasomal degradation pathway and is essential for Ad replication (Figure 1) [38–42] . Currently, it is becoming increasingly apparent that Ads possess additional strategies to target host cell proteins via additional Cullindependent E3 ubiquitin ligase complexes. Recent reports have provided evidence that E1B55K-dependent/E4orf6-independent as well as E1B-55K-independent/E4orf6-dependent, Cullin-based E3 ubiquitin ligase activities neutralize host defense processes (Figure 2) [43,44] . In line with this, Cheng and coworkers recently future science group

found that E3 ligase complexes are assembled by different Ad serotypes. These findings indicate that different Ads exhibit distinct Cullinspecificities and therefore target a different selection of respective cellular substrates [45,46] . In this context it is reasonable to mention that excellent work by Lethbridge and coworkers demonstrated that absence of viral early gene product E4orf6 significantly enhanced E1B-55K matrix association in the nucleus as well as E1B55K SUMO modification [47] . In summary, these results indicate that SUMOylation of E1B-55K might play an essential role in degrading cellular targets, although the contribution of E1B-55K or E4orf6 posttranslational modifications on Ad-induced proteolysis is far from understood. www.futuremedicine.com

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Review


E1B-55K

E1B-55K

E4orf6

E4orf6

Mre11

Integrin α3

Daxx

Mre11

Rad50 Rad50 Nbs1

p53

TopBP1

BLM MRN complex DNA ligase IV Cell cycle regulation, apoptosis

Rbx1 Elongin B/C Cullin 2/5

Modulation of cell surface receptors

DNA–double strand break repair/NHEJ, cell cycle checkpoint signaling

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Chromatin structure, transcriptional repression

Proteasomal degradation

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Ubiquitin Ubiquitin Ubiquitin Cellular E3 ubiquitin ligase components

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Efficient adenovirus replication and progeny production

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Figure 2. Overview of adenovirus-mediated ubiquitin/proteasome dependent modulation of different host cell responses. Adenoviruses encode the viral components E1B-55K and E4orf6, which assemble a functional E3 ubiquitin ligase complex, together with cellular factors mentioned in the text. This figure highlights the functional consequences of adenovirus-dependent proteasomal degradation of so far known cellular targets, leading to efficient viral replication and progeny production. NHEJ: Nonhomologous end joining.

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Taken together, these data emphasize that the ongoing detection of new viral substrates, as well as identification of the underlying processes by which different Ad serotypes modulate host cell pathways will be important for gaining further insights into how these pathways operate. The variability of ligase components seems to be a general principle of multimeric E3 ligases and indicates a much more universal phenomenon than is currently appreciated for Ad-dependent protein degradation. However, in order to better understand the mechanistic strategies of each multimer, more data are required. Systematic elucidation of substrate preferences in the context of infection with each Ad serotype, determination of ubiquitin chain type in the context of appropriate E2 enzymes by mass spectroscopic

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techniques and identification of regulatory subunits, including their contribution to the activity of these enzymes is still lacking. Fundamental understanding of these complex biological machines will allow us to focus targeting strategies in drug design research in the context of various diseases in which they participate. Adenovirus-dependent degradation of proapoptotic factors p53

Many viruses including Ad have acquired functions that induce metabolic activity, promote cell cycle progression, and prevent cellular apoptosis and immune responses, thus establishing a replication competent milieu in the host cell. After entry into the cell, viral genomes and modulation of future science group


Review

Box 2. Formation of adenoviral E3 ubiquitin ligase complexes. Elongins B and C are components of the von Hippel Lindau (VHL) complex [155] Elongin B/C interact with various other proteins via a BC-Box [155,156] Elongin B/C interact with adenovirus (Ad) E4orf6 via two functional BC-boxes Rbx1 (ROC1) protein is a component of both, the SCF (Skp1-Cullin1/Cdc53-F-box) and VHL tumor suppressor complexes [157] Ad assemble an E3 ubiquitin ligase complex of E1B-55/E4orf6, Cullin family members 2/5, Elongin B/C and the RING protein Rbx1 Ad E1B-55/E4orf6 E3 ubiquitin ligases are Cullin2/5-based, similar to VHL and SCF E3 ubiquitin ligase complexes belonging to RING-type E3 ligases E4orf6 is linked to Cullin2/5 via Elongin B/C E4orf6 selects the Cullin specifically, as Elongins B/C can be assembled with either Cullin2 or Cullin5 Cullin specificity and substrate is dependent on the Ad serotype (see Table 1) More than 50% of E1B-55K in Ad-infected cells associates with the Ad E3 ligase complex E1B-55K is the substrate recognition unit in Ad-infected cells and therefore associates with the cellular substrate

Data taken from [6,36–38,46,154].

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PML-NBs are host cell nuclear structures that are targeted by DNA viruses early after infection and are sites of active viral gene transcription [64–67] . Recent evidence suggests that PML-NBs and associated factors contribute to antiviral defense mechanisms. During Ad infection, viral genomes colocalize with PML-NBs and replication centers arise in close proximity to these cellular structures [68–70] . A track-like rearrangement of PML-NBs occurs in response to expression of the Ad E4orf3 polypeptide [13,71,72] . Recently, the PML-NB associated Daxx protein was identified to significantly repress Ad replication [44] . Daxx is a ubiquitously expressed protein that modulates apoptotic pathways and basal transcription [73,74] . In the presence of E1B-55K, Daxx is depleted from the PML-NBs and is dynamically redistributed between E1B55K/p53 protein complexes and PML-NBs [75] . Furthermore, Ad E1B-55K was identified as a viral interaction partner of Daxx [44,75,76] and required for proteasomal degradation of Daxx during Ad infection [44,77] . Remarkably, in contrast to already mentioned cellular targets of Ad E3 ubiquitin ligases, E4orf6 is dispensable for Daxx reduction (Figure 1B) . This indicates that proteasomal degradation of Daxx is likely to be independent of the activity of the E1B-55K/ E4orf6 E3 ligase complex. Recent studies suggest that Daxx reduction occurs via a proteasomal E1B-55K-/Cullin dependent mechanism [44] . These data indicate that E1B-55K is sufficient to assemble cellular components into a functional ubiquitin ligase complex. In fact, a highly conserved BC-Box motif, known to mediate the interaction with Elongin B and C, containing the consensus sequence is present in the adenoviral E1B polypeptide, although it seems to be

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diverse cellular signaling cascades induce danger signals prior to activation of the host DNA damage responses. Normally, these processes culminate in cell cycle arrest or induction of apoptotic pathways. Thus, to secure propagation and proper replication, Ad-expressed early E1A gene products stabilize the tumor suppressor p53 and other cellular factors [48] . However, since coexpression of the early Ad E1B-55K protein in turn modulates p53 activity, accumulation of this apoptotic factor does not elicit programmed death [49–54] . E1B55K has been demonstrated to interact with p53 [55,56] , to promote its transcriptional repression [49,52,53] and to drive its nuclear-cytoplasmic relocalization [57,58] , thereby abrogating p53 functions and generating antiapoptotic pressure. Interestingly, E4orf6 was also shown to inhibit p53-mediated transcriptional activation both in vitro and in vivo [59] . To ensure proper Ad replication, p53 is proteolytically degraded by the E3 ubiquitin ligase assembled with E1B-55K and E4orf6 (Figure 2) [38,60,61] . Ubiquitination of p53 was assessed by the appearance of slower migrating forms of p53, only seen when E1B55K and E4orf6 were present [38] . The degradation of p53 prevents the early apoptotic death of infected cells and permits a productive replication cycle. In this context it is reasonable to mention that E1B-55K is a substrate for the host cell SUMO modification pathway [57,58,62] and acts as a SUMO E3 ligase, inducing the SUMOylation of p53 in vitro as well as in vivo [63] . This results in inactivation of p53 protein via restriction to promyelocytic leukemia nuclear bodies (PMLNBs) [63] . In summary, these data indicate that E1B-55K is connected to the SUMOylation system of the host cell, and as yet we can only speculate about any functional consequences for protein degradation and/or E3 ligase formation [62] . future science group

www.futuremedicine.com

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Table 1. Host cell target proteins for human adenoviruses. kDa

Major function in host cell

Ad proteins

Ad serotype†

p53

53

DNA repair Apoptosis Cell cycle regulation Transcriptional regulation

E1B-55K E4orf6

Ad 12 (A), Ad 5 (C), Ad 40 (F)

Mre11

80

DNA repair Homologous recombination Telomere maintainance 3´– 5´ exonuclease activity

E1B-55K E4orf6 (E4orf3)

Ad 12 (A), Ad 16 (B1), Ad 34 (B2), Ad 5 (C), Ad 40 (F), Ad 9 (D)

DNA ligase IV

109

ATP-dependent DNA ligase DNA repair V(D)J recombination

E1B-55K E4orf6

Ad 12 (A), Ad 16, 3, 7 (B1), Ad34, 11 (B2), Ad 5 (C), Ad 9 (D), Ad 4 (E), Ad40 (F)

BLM

159

DNA repair ATP-dependent DNA Helicase Homologous recombination

E1B-55K E4orf6

TopBP1

173.3

DNA repair DNA topoisomerase-binding protein

E4orf6

Integrin a3

130

Cell receptor Cell signaling Cell adhesion

Daxx

120

Apoptosis Antiviral defense Transcriptional regulation Chromatin remodeling

Ref. [36,38,45,46,144]

[40,45,46,86,91]

[39,45,46,86,153]

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Protein

[41]

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Ad 5 (C)

[43,46]

E1B-55K E4orf6

Ad 12 (A), Ad 5 (C), Ad 4 (E), Ad 40 (F)

[42,45,112]

E1B-55K

Ad 5 (C)

[44,75–77]

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rP

Ad 12 (A),

For each protein, predicted molecular weight in kDa is indicated. Only Ad serotypes tested so far are included. Ad: Adenovirus.

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†

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nonfunctional [15] . In line with this, E1B-55K additionally contains a cysteine/histidine-rich region, which contains several domains partly resembling the consensus motifs for RING fingers and might be capable of catalyzing ubiquitin transfer to substrates [35,78] . It seems reasonable that Daxx proapoptotic functions could be inhibited by interaction with the antiapoptotic E1B-55K protein and its subsequent degradation. Therefore, functional inactivation of Daxx could be considered to be a basic step in Ad-mediated prevention of cell-death. Furthermore, it was shown that E1B-55K dependent Daxx degradation is required for efficient transformation of primary mammalian cells in vitro. An E1B-55K mutant lacking the SUMO-1 conjugation site (SCS) was sufficient for Daxx interaction, but was not able to initiate E1B-55K dependent proteasomal degradation of this cellular factor [77] . These

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results together with the observation that E1B55K SUMOylation is required for efficient transformation provides evidence for the idea that SUMO-1 conjugated E1B-55K-mediated degradation of Daxx plays a key role in adenoviral oncogenic transformation. In line with this, further E1B-55K mutants lacking the ability to degrade Daxx, show no transforming potential [77] . Moreover, these findings substantiate the hypothesis that mechanisms for efficient adenoviral transformation of primary cells can be separated from those required for the inhibition of p53-stimulated transcription. Adenovirus-dependent degradation of cellular DNA repair proteins Mre11

Host DNA damage response (DDR) and repair machinery impose a huge hurdle to the viability and propagation of DNA viruses. To counter future science group


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significantly reduced in the presence of E1B-55K and E4orf6 via the Cullin-based E3 ubiquitin ligase complex (Figure 2) . Similar to Mre11, reduction of DNA ligase IV is dramatic when E4orf6 levels are increased [39] . It is known that DNA ligase IV complexes with the XRCC4 adapter protein to catalyze the rejoining step in NHEJ [95] . DNA ligase IV was further identified to be recruited by DNA-PK and is integral for V(D)J recombination to generate diverse immune system components such as immunglobulins and T-cell receptors. In addition, DNA-PK is described to recruit several other repair proteins, including the Mre11/Rad50/NBS1 complex [96] . The fact that Mre11 and DNA ligase IV are both targeted for proteasomal degradation by E1B-55K and E4orf6 confirms that Ad have acquired multiple independent strategies to inhibit genome concatemerization. Since DNA ligase IV is essential for NHEJ, Ad infection results in globally inhibiting cellular NHEJ repair responses. In the line with these findings it has been demonstrated that concatemerization does not occur in human cells lacking DNA-PK. Substantiating these observations, E4orf3 and E4orf6 have been reported to interact with DNA-PK [84] . Therefore, Ads have become associated with several processes that disrupt the cellular NHEJ pathway, allowing viral DNA packaging in the late phase of infection as well as efficient replication and progeny production.

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this, several DNA viruses have acquired early viral genes that degrade or target key cellular factors of the DDR and repair machinery (Figure 2) . The recognition and repair of DNA doublestrand breaks (DSBs) is a complex process mediated by a multitude of regulatory factors. The MRN complex (Mre11, Rad50 and Nbs1) acts as a sensor in this cascade, recognizing DSBs and subsequently activating ATM, which phosphorylates several downstream proteins involved in cell cycle control and DNA repair [79–82] . Initial reports showed that in contrast to wild-type Ad, infections with mutant Ad containing deletions in the E4 region resulted in intense activation of ATM/ATR mediated DNA damage response pathways [40,83] . In addition, the mutant viral genomes formed substantial concatemers owing to the activation of DNA repair mechanisms [40,84,85] . As a consequence of concatemerization, Ad genomes are too large to be packaged, leading to abortive infections. These data indicate that the E4 early proteins are crucial for suppressing the host DDR and thereby preventing viral concatemerization. The Ad E1B-55K/E4orf6 E3 ubiquitin ligase complex targets Mre11 for degradation, further protecting the viral genome integrity (Figure 2) . In the absence of Mre11, Rad50 and NBS1 become unstable, independent of a viral infection [37,86] . The Ad-mediated inhibition of MRN by the E3 ubiquitin ligase complex thereby acts to block concatemer formation and DNA damage signaling, and therefore allows efficient virus growth [40] . E4orf3 is another E4 gene product that shares functional redundancies with E4orf6 [14,87] . E4orf3 has similarly been shown to inhibit genome concatemerization and activation of DNA repair mechanisms by redistributing the MRN complex into nuclear tracks and cytoplasmic aggresomes [40,88–91] . Taken together, Ad have acquired the capacity to aggressively inhibit MRN activity, by either proteasomal degradation of Mre11 via the E1B-55K/E4orf6 E3 ubiquitin ligase complex or E4orf3 dependent relocalization of MRN into cytoplasmic inclusions, thus resulting in productive infection.

Review

DNA ligase IV

Nonhomologous end joining (NHEJ) is the predominant DSB repair pathway of cells in G1 and S-phase and comprises several factors [92–94] . Recently published results show that the protein levels of DNA-PK, Ku70, Ku80, XRCC4, XLF and Artemis are not affected during the course of Ad infection. By contrast, DNA ligase IV is future science group

Bloom’s helicase

In the light of the above findings, considerable effort has been directed towards unraveling Ad modulation of cellular DNA repair mechanisms. Recent studies of Orazio and coworkers identified Bloom’s helicase (BLM) as being proteasomally targeted by the Ad E3 ubiquitin ligase complex in the presence of both early viral proteins E1B-55K and E4orf6 (Figure 2) [41] . Efficient and precise elimination of DNA damage require a large number of factors [97,98] . As mentioned above, prior to enzymatic processing, the MRN complex senses DNA breaks. Subsequently, the cell recruits helicases and nucleases to restrict DNA ends and promote repair [98,99] . BLM is implicated in DNA endprocessing and belongs to the RecQ helicases, known to maintain genome integrity [100–102] . In contrast to BLM, other family members of the RecQ helicase family (WRN, RecQ1, RecQ4 and RecQ5) were completely unaffected by the presence of E1B-55K and E4orf6 [41] . The functional relevance of BLM degradation by Ad www.futuremedicine.com

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remains unclear, since it shows no capacity to promote viral genome concatemerization [41] . However, it was proposed that BLM may play a positive role in early steps of Ad infection, since the protein locates adjacent to viral replication sites in the host cell nucleus [41] . TopBP1

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TopBP1, the topoisomerase IIβ-binding protein is involved in DNA damage response and cell cycle checkpoint control [103,104] . TopBP1 is a substrate of ATM kinase and is phosphorylated rapidly after irradiation. Inhibition of TopBP1 expression was further shown to induce apoptosis, indicating that TopBP1 function may be required for cell survival [105] . Recently, it was shown that Ads also target TopBP1, thereby challenging ATR signaling [43] . In contrast to Mre11, DNA ligase IV and BLM elimination, TopBP1 is specifically degraded by Ad type 12 E4orf6 protein, whereas Ad5 has no measurable effect (Figure 1B) [43] . Further studies showed that Ad12 E1B-55K is not relevant for the Cullin2-/E4orf6-dependent proteasomal degradation of TopBP1 [43] . It has been proposed that Ad12-mediated inhibition of TopBP1 interferes with ATR-dependent phosphorylation of Chk1, leading to the modulation of ATR signaling and DNA damage response [43] . Thus, in contrast to cells expressing Ad5 E4orf6, Ad12 E4orf6 selectively blocks activation of the ATR/ Chk1 signaling pathway. Taken together, these findings identify unique strategies acquired by different serotypes to target the DDR and inhibit antiviral measures in host cells.

identified as a substrate of the E1B-55K/E4orf6 E3 ubiquitin ligase complex [42,112] . This integrin comprises a3 and b1 subunits and localizes to the cell surface or within intracellular compartments [113,114] . Integrin a3 is ubiquitously expressed in different tissues, where it binds to fibronectin, collagen, thrombospondin1 and laminins [115] . A significant decrease in integrin a3 protein levels was observed when E1B-55K and E4orf6 were coexpressed from viral vectors [42] . However, transfection of plasmid DNA expressing the E1B-55K and E4orf6 protein resulted in a very modest reduction of integrin a3. This observation indicates that efficient integrin a3 degradation requires cells to be infected with Ads. Consistent with p53, Mre11, BLM and DNA ligase IV, integrin a3 is similarily targeted by the viral E3 ubiquitin ligase and degraded via a Cullin5 dependent mechanism, since dominantnegative Cullin5 mutants prevented integrin a3 proteasomal degradation [42] . In uninfected human H1299 cells, integrin a3 localizes in a diffuse intracellular pattern, concentrated at the plasma membrane and in small perinuclear bodies, suggested to correspond to perinuclear recycling centers of membrane receptors. In Ad5 wild-type infected cells integrin a3 was detected to colocalize with E1B-55K in aggresomes, surrounded by a vimentin cage. In these structures E1B-55K is believed to trap the integrin, thus preventing its recycling to the plasma membrane and enhancing its degradation by the E4orf6/ E1B55K complex. In this context, no direct E1B55K/integrin a3 interaction was identified, suggesting that further studies are needed to examine the class of cell surface proteins. It has already been demonstrated that degradation of integrins initiates apoptotic pathways [116] . Therefore, it is tempting to speculate that Ad-dependent inhibition of integrin a3 enhances cell lysis, leading to efficient particle release [42] . Together, these recent observations provide compelling evidence for how Ads affect cell–cell interactions to improve virus spreading. Cell surface molecules therefore represent important new targets for degradation by the E1B-55K/E4orf6 E3 ubiquitin ligase complex.

Adenovirus-dependent degradation of cellular receptor proteins Integrin a3

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Recently, the E4orf6/E1B55K E3 ubiquitin ligase complex was shown to be involved in modulating cellular surface proteins, a function believed to mediate Ad spreading in the host organism. Efficient internalization of Ad into cells is initiated by the viral fiber binding to the CAR or CD46 [106] . Thus, interaction of viral penton base polypeptides with cell surface integrins triggers virus internalization via clathrin-coated pits [107,108] . To date, more than 20 integrin members, composed of a and b subunit heterodimers, have been identified. Depending on the ligand specificities, integrins are involved in cell adhesion, proliferation, differentiation, translation, migration and apoptosis [109–111] . Integrin a3 is an alternative Ad receptor and was recently

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Adenovirus-dependent degradation of adeno-associated virus capsid proteins

Adeno-associated viruses (AAVs) belong to the nonenveloped parvoviridae family. Productive AAV replication was shown to be mediated by Ad, Herpes simplex viruses or toxic agents future science group


viral infection [131] . It has been postulated that proteolysis of cellular structural proteins such as lamin B1 by caspases likely cooperates with autophagy vacuolization to destroy host cell structure and promote the release of new viral particles. Therefore, unlike lysosomal hydrolases, caspases play an integral role in cell lysis during Ad infection [133] . In summary, autophagy plays an essential role in Ad-mediated cell lysis by regulating caspase activity. These findings are also of clinical relevance since new mechanisms for Ad-induced cell lysis may improve oncolytic strategies in cancer therapy.

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Adenovirus-dependent degradation by VA-RNAs

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miRNAs are small, noncoding, ssRNAs of 19–24 nucleotides that regulate gene expression at the post-transcriptional level [134] . Hence, alterations in miRNA expression can strongly influence cellular homeostasis and are therefore linked to human cancer and neurodegenerative diseases [135] . In the nucleus, pri-miRNA transcripts containing hairpin structures are cleaved by Drosha and RNA-binding partners. The resulting processed stem–loop structure (pre-miRNA) is recognized by exportin 5 [136–138] and transits to the cytoplasm, where it is then cleaved by Dicer. The guide strand of the miRNA, generated by Dicer is incorporated into the RISC complex, containing the Argonaute proteins. Argonaute-bound miRNA specifically recognize cellular mRNAs to either induce their degradation and/or inhibit their translation [139] . Recently, Bennasser and coworkers described a previously unknown cross-regulation between pre-miRNA and Dicer. Overexpression of premiRNA was shown to decrease Dicer levels, suggesting a mechanism that contributes to the homeostatic control of miRNA biogenesis [140] . In line with this, knockdown of Dicer led to an increased level of Drosha by an unknown mechanism. Most likely, regulation of Dicer may have been acquired by viruses to modulate cellular miRNA expression and overcome RNAi. Indeed, in vitro assays and cellular RNA immunoprecipitation experiments showed that exportin 5, the karyopherin responsible for pre-miRNA nuclear export, interacts directly with Dicer mRNA. During Ad5 replication, VA1- and VA2-RNA are produced in large amounts and accumulate in infected cells, reaching up to 20% of cellular RNA [141] . Titration of exportin 5 by overexpression of either pre-miRNA or VA1-RNA of Ad5

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(e.g., UV and hydroxyurea) [117–121] . Five Ad gene products, E1A, E1B-55K, E4orf6, E2A and VA-RNA are required for synthesis of new AAV particles [117,122] . Since two AAV proteins are the only viral gene products known so far to be targeted by the Ad E3 ubiquitin ligase complex it is worth including these observations in this article. Hence, it was shown that AAV5 small Rep52 and capsid proteins were degraded in the presence of Ad E1B-55K and E4orf6 in a Cullin5 dependent manner via the ubiquitin/proteasomal pathway [123,124] . Furthermore, E1B-55K/ E4orf6 dependent degradation of Mre11 was recently shown to enhance AAV2 replication [125] . Additionally, Ad E4orf6 is required for the conversion of AAV type 2 (AAV2) genomic ssDNA into dsDNA replication intermediates [125,126] . When considered together, these findings suggest that degradation of AAV5 by E1B55K and E4orf6 supports Ad replication and impairs AAV gene expression during Ad/AAV coinfection. This function of Ad further represents an important regulator in AAV biology and should be considered in AAV-based gene therapy approaches.

Review

Adenovirus-dependent degradation by autophagy

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Autophagy is a dynamic eukaryotic process that induces the degradation of long-lived cytoplasmic proteins and damaged organelles [127] . This process involves the sequestration of cytoplasmic regions into vesicles (autophagosomes), followed by enzymatic digestion of the engulfed factors prior to lysosomal fusion [128] . The formation and maturation of autophagosomes requires the activation of two ubiquitin-like molecules, LC3 and ATG12 by the activating enzyme ATG7 [127] . Autophagy not only participates in the maintenance of cellular homeostasis, but also plays a crucial role during viral infections [129–131] . Recently, it was demonstrated that the destruction of cellular structures at the end of the Ad infectious cycle, and prior to the release of viral progeny, is mediated by autophagy-dependent membrane reorganization [132] . During Ad infections the ATG12–ATG5 complex is upregulated, implicating that autophagy may functionally contribute to a productive Ad lifecycle. Moreover, it has been shown that caspases are activated during Ad-induced autophagy and mediate cell lysis. The observation that cytochrome C is not released into the cytoplasm after Ad infection indicates that the extrinsic pathway is responsible for caspase activation at the late stage of future science group


9

Review


resulted in loss of Dicer mRNA/exportin 5 interaction and reduction of Dicer protein levels [140] . This downmodulation correlates with increased amounts of VA1-RNA of Ad5. This saturation also occurred during Ad5 infection and enhanced viral replication [140] . Together, the results of this study clearly show that Ad5 VA-RNAs, massively produced at the late stage of Ad5 infection, are able to restrict Dicer expression by saturating exportin 5 and preventing nuclear export of Dicer

mRNA. In conclusion, this may illustrate a new gene-regulatory mechanism acquired by adenoviruses, involving degradation of cellular proteins by modulating mRNA export. Conclusion & future perspective

It is irrefutable that viruses have acquired functions that target host cell signaling pathways leading to their continued propagation. In the context of productive infection, Ad functions neutralize

Executive summary Adenovirus-dependent E3 ubiquitin ligases Adenoviruses (Ad) have acquired strategies to modulate double-strand break repair, apoptosis, gene expression and host cell immune responses, resulting in efficient virus replication. Ad E3 ubiquitin ligase is a high molecular mass complex (800–900 kDa), found to be essential for Ad replication by sequestering specific cellular substrates into a proteasomal degradation pathway to neutralize host defense mediated processes. E4orf6 connects E1B-55K to an E3 ubiquitin ligase containing Rbx1/ROC1/Hrt1, Cullin2/5, Elongin B and C, similar to the SCF and VBC complexes, which recruit Cullin1 and Cullin2. E1B-55K serves as the substrate recognition unit, while E4orf6 assembles the cellular components of the complex.

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Adenovirus-dependent degradation of proapoptotic factors Ad E3 ubiquitin ligase complex degrades p53, preventing apoptotic stimuli and host cell death mechanisms. Promyelocytic leukemia nuclear bodies and associated factors contribute to antiviral defense mechanisms of the host cell. Promyelocytic leukemia nuclear bodies-associated Daxx protein was found to significantly repress Ad replication. Daxx protein concentrations are reduced during lytic Ad infection. Ad-mediated proteasomal degradation of Daxx is independent from the activity of the E1B-55K/E4orf6 E3 ligase complex and occurs via a proteasomal E1B-55K/Cullin-dependent mechanism.

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Adenovirus-dependent degradation of cellular DNA repair proteins Mre11, DNA ligase IV, BLM and TopBP1, described as cellular proteins with various functions during DNA repair, are identified as cellular substrates of the Ad E1B-55K/E4orf6 E3 ligase complex. Importantly TopBP1 is only degraded during Ad12 infection in a so far unknown E4orf6-dependent, E1B-55K-independent and Cullin2-mediated proteasomal pathway.

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Adenovirus-dependent degradation of cellular receptor proteins E4orf6/E1B55K E3 ubiquitin ligase complex was shown to be involved in modulating cellular surface proteins to mediate Ad spreading in the host organism. Integrin a3, known to be an alternative Ad receptor was recently identified as a substrate of the Ad E1B-55K/E4orf6 E3 ubiquitin ligase complex.

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Adenovirus-dependent degradation of AAV capsid proteins Productive replication of Adeno-associated virus (AAV), a nonenveloped parvovirus, was shown to require Ad E1A, E1B-55K, E4orf6, E2A and virus-associated (VA) RNA gene products. AAV5 small Rep52 and capsid proteins were degraded in the presence of Ad E1B-55K and E4orf6 by a Cullin5 containing E3 ubiquitin ligase via the ubiquitin/proteasomal pathway. AAV replication requires functional Ad mediated E3 ubiquitin ligase activity.

Adenovirus-dependent degradation by autophagy Autophagy induces degradation of long-lived cytoplasmic proteins and damaged organelles. Formation and maturation of autophagosomes requires activation of LC3 and ATG12 by ATG7. Destruction of cellular structures at the end of Ad infectious cycles, prior to release of viral progeny is mediated by autophagy-dependent membrane reorganization and caspase activation.

Adenovirus-dependent degradation by VA-RNAs Overexpression of pre-miRNA decreases Dicer levels, suggesting a mechanism that contributes to the homeostatic control of miRNA biogenesis. Knockdown of Dicer leads to increased levels of Drosha by an unknown mechanism. Exportin 5, the karyopherin responsible for pre-miRNA export, interacts directly with Dicer mRNA. Titration of exportin 5 by overexpression of either pre-miRNA or Ad5 VA1-RNA results in loss of Dicer mRNA/exportin 5 interaction and reduction of Dicer protein levels.

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late mRNAs from the nuclear compartment to the cytoplasm, while simultaneously repressing cellular mRNA export [147] . It is still not well understood how the E1B-55K/E4orf6 E3 ligase complex mediates the exclusive nuclear export of viral late mRNAs [6,15] . However, it is speculated that the Ad E3 ligase complex initiates degradation of a so far unknown cellular mRNA export factor or essential RNA binding factors (e.g., hnRNPs) [6] . This would result in failure of cellular mRNA export and enhance Ad survivability and pathogenesis. As mentioned above, chromatin-remodeling factors such as Daxx, being part of the SWI/ SNF complex, are degraded due to Ad infection [44,148,149] . This raises the question of whether Ad, like many other DNA viruses, encode for proteins capable of manipulating the epigenetic environment of the host, thus promoting viral propagation and rendering the host immune response inactive [150–152] . Immune evasion strategies acquired by viruses are of major concern in modern biomedical research, and epigenetic modulation is emerging as a valid viral trait. Taken together, the detailed molecular mechanisms and repertoire of cellular targets remain elusive, necessitating future studies to unravel the complete program of Ad-mediated E3 ubiquitin ligase complexes.

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host-defense processes to promote viral replication (Figure 2 & Table 1) . Furthermore Ad gene products manipulate cellular destruction pathways, first to prevent apoptosis prior to viral genome amplification and later to ensure the release of viral progeny (Table 1) . Ongoing investigations of the cellular pathways modulated by Ad will be of great interest, particularly since Ad-based vectors frequently serve as tools in human gene therapy. Ad constitute an ideal model system to analyze the transforming capabilities of small DNA tumor viruses, as well as the underlying molecular principles of virus-induced tumorigenesis. Recent reports have suggested that Ad-mediated degradation of host cell substrates contributes to the oncogenic properties of early Ad gene products. Consistent with this, E1B-55K oncoproteins are capable of completely transforming nonpermissive host cells by restricting apoptosis and cellular growth arrest in combination with E1A expression [57,142] . Inhibition of p53 plays a key role in this process [58,60,63,143–145] . Nevertheless, additional functions of E1B-55K and interactions with cellular DNA repair, transcription and apoptosis factors including Mre11 and Daxx also contribute to the transforming potential of E1B-55K. E1B-55K mutants, which lose the ability to degrade Daxx/p53 or to interact with Mre11 show reduced transforming potential in primary rodent cells [75–77,146] . These data indicate that efficient E1B-55K transformation requires the successful hijacking of the host ubiquitin/proteasome system and Ad-mediated recruitment of cellular substrates that normally protect the integrity of the cell. The Ad E3 ubiquitin ligase complex, assembled by E1B-55K and E4orf6, is essential to shut down host cell antagonizing processes. It is well established that both early viral proteins are required for the preferential transport of viral

Review

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Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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