The interplay between host and viral factors in shaping the ... - Nature

Immunology and Cell Biology (2007) 85, 46–54 & 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00 www.nature.com/icb

REVIEW

The interplay between host and viral factors in shaping the outcome of cytomegalovirus infection Anthony A Scalzo1,2, Alexandra J Corbett1,2, William D Rawlinson3, Gillian M Scott4 and Mariapia A Degli-Esposti1,2 Cytomegalovirus (CMV) remains a major human pathogen causing significant morbidity and mortality in immunosuppressed or immunoimmature individuals. Although significant advances have been made in dissecting out certain features of the host response to human CMV (HCMV) infection, the strict species specificity of CMVs means that most aspects of antiviral immunity are best assessed in animal models. The mouse model of murine CMV (MCMV) infection is an important tool for analysis of in vivo features of host–virus interactions and responses to antiviral drugs that are difficult to assess in humans. Important studies of the contribution of host resistance genes to infection outcome, interplays between innate and adaptive host immune responses, the contribution of virus immune evasion genes and genetic variation in these genes to the establishment of persistence and in vivo studies of resistance to antiviral drugs have benefited from the well-developed MCMV model. In this review, we discuss recent advances in the immunobiology of host–CMV interactions that provide intriguing insights into the complex interplay between host and virus that ultimately facilitates viral persistence. We also discuss recent studies of genetic responses to antiviral therapy, particularly changes in DNA polymerase and protein kinase genes of MCMV and HCMV. Immunology and Cell Biology (2007) 85, 46–54. doi:10.1038/sj.icb.7100013; published online 5 December 2006 Keywords: viral infection; genetic variability; immune evasion; cytomegalovirus; NK cells; CTL

The outcome of host–virus interactions is determined by a range of factors including inherent host resistance and environmental factors, such as nutritional status, together with viral factors such as variation in genes governing levels of infectivity, tropism and immune evasion. Some host genes that confer resistance are polymorphic between individuals within populations, and thus different individuals will vary in their relative susceptibility to infection. The outbred nature of the human population has made it difficult to assess the contribution of host resistance genes in determining disease outcome of human cytomegalovirus (HCMV) infection. However, murine CMV (MCMV) infection of inbred mouse strains has provided many important insights into host genes that regulate CMV infection. ROLE OF HOST RESISTANCE GENES IN CONTROLLING CMV INFECTION Research on the contribution of host resistance genes to the outcome of MCMV infection was initiated about three decades ago.1,2 Studies by Grundy et al.3 established that both murine major histocompatibility complex (MHC) or H2 loci, together with non-H2 loci contribute to the control of acute MCMV infection. Resistance was associated with the protective H2k haplotype. Non-H2 resistance loci

were identified as being present on the C57BL/6J (abbreviated B6), C3H and CBA genetic backgrounds. Role of H2 genes in resistance To investigate the contribution of the H2 complex to MCMV resistance, Price et al.4 isolated macrophages from mice differing in H2 genotype and assessed their susceptibility to MCMV infection in vitro. These studies showed that cells from H2k haplotype mice were considerably less sensitive to infection,4 and mapping studies using intra-H2 haplotype mice indicated that MHC class I genes contributed to this effect.5 Further evidence for a role of MHC class I molecules was demonstrated by the finding that transfection of cells that were largely resistant to MCMV infection with specific MHC class I molecules increased their susceptibility to MCMV infection.6 Altogether, these data indicated that MHC class I molecules could function as a receptor or coreceptor for MCMV entry. Subsequent investigations using MHC class I-negative, b2-microglobulin (b2m) knockout mice on the B6 background,7 or 129/SvB6 background,8 did not support a role for class I molecules as entry coreceptors. However, it is conceivable that these analyses may have missed differences between wild-type and b2m / mice. The in vivo assessments of virus replication were only performed in the spleen7 or

1Immunology and Virology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Western Australia, Australia; 2Centre for Experimental Immunology, Lions Eye Institute, Nedlands, Western Australia, Australia; 3Department of Microbiology, Virology Division, SEALS, Prince of Wales Hospital, Randwick, New South Wales, Australia and 4Virology Research, POWH and UNSW Research Laboratories, Prince of Wales Hospital, Randwick, New South Wales, Australia Correspondence: Dr AA Scalzo, Centre for Experimental Immunology, Lions Eye Institute, 2 Verdun St, Nedlands, Western Australia 6009, Australia. E-mail: [email protected] Received 9 October 2006; accepted 12 October 2006; published online 5 December 2006

The interplay between host and viral factors AA Scalzo et al 47

after the peak of visceral virus replication (i.e. after 2 weeks postinfection (p.i.)).8 The in vitro demonstration of H2 effects in fibroblasts is dependent on seeding densities of fibroblasts cultures.9 Hence, further studies on the role of MHC class I molecules as coreceptors for MCMV entry are warranted using b2m / mice on the BALB/c background where the contribution of natural killer (NK) cells to resistance is not as great. More recently, a role for the protective H2k haplotype in contributing to NK cell-associated resistance in the MA/My mouse strain has been established10,11 which is discussed below. Effects of host genotype on type I interferon production Type I interferons (IFN) are among the key early cytokines that have direct antiviral as well as immunoregulatory effects (reviewed by Biron12), including the regulation of NK cell cytolytic activity. Analyses of IFN levels in sera at 6 h p.i. have revealed that mousestrain-dependent differences exist and that these are dependent on non-H2 genetic effects.13 A more recent study by Dalod et al.14 confirmed non-H2 regulation of IFN-a production in H2b-matched mice, with 129/J mice producing 47-fold more than B6 mice at 36 h p.i. Whether the strain-dependent differences in levels of type I IFNs following MCMV infection are due to different frequencies of cells that produce this cytokine, such as plasmacytoid dendritic cells (DCs), or allelic variability in genes that regulate type I IFN production, remains to be defined. Host genes that regulate NK cell control of MCMV infection The importance of NK cells in controlling MCMV was demonstrated about two decades ago in studies showing exacerbation or amelioration of infection when adult B6 mice were depleted of NK cells or when NK cells were adoptively transferred into suckling mice before MCMV infection, respectively.15,16 Analyses of NK cell activity in inbred strains following MCMV infection also revealed a general correlation between the level of NK cell cytotoxicity and resistance status to MCMV.17 Cmv1. A Mendelian analysis of the genetic basis for differences in the level of MCMV replication in the spleens of susceptible BALB/c mice and moderately resistant B6 mice led to the identification of the Cmv1 resistance locus. Cmv1 was shown to have a dominant effect and was mapped to the distal region of mouse chromosome 6.18 The effect of Cmv1 was subsequently shown to be mediated via NK cell control of viral infection and the locus was mapped to the NK cell gene complex (NKC), a region which encodes both inhibitory and activation NK cell receptors.19 Following detailed genetic and physical mapping studies, the gene mediating the Cmv1 effect was identified as Ly49h or Klra8, a member of the Ly49 family of molecules in the distal NKC region.20–22 Ly49H is an activation receptor expressed on NK cells. Although Ly49H lacks intracellular signaling motifs in its cytoplasmic domain, it physically associates with the DAP12 adaptor molecule via residues in its transmembrane domain and phosphorylation of DAP12, via the immunoreceptor tyrosine-based activation motif (ITAM) present in the cytoplasmic domain of this molecule, is responsible for NK cell signaling.23,24 Formal proof of the role of Ly49H in mediating the Cmv1 effect came from the finding that Ly49H transgenic mice on the susceptible BALB/c and FVB genetic backgrounds are resistant to MCMV infection.25 The critical role of the DAP12 adaptor in Cmv1/Ly49H-mediated resistance in B6 mice was independently confirmed by studies showing that mice carrying targeted mutations in the DAP12 ITAM signaling motif are highly susceptible to MCMV infection.26

Analyses of how Ly49H specifically recognizes MCMV-infected cells and triggers NK cell activation revealed that it binds to a MHC class I-like, MCMV-encoded molecule called m157.27,28 Engagement of m157 is sufficient to trigger NK cell cytolysis as well as the secretion of IFN-g and the chemokine ATAC/lymphotactin.28 Also it binds the Ly49H activation receptor that is expressed in B6 mice, the m157 viral glycoprotein also binds to the inhibitory Ly49I molecule expressed in the 129/J mouse strain,27 raising the possibility that, at least in some infections, m157 may function as an immune evasion molecule. Disruption of the m157 gene in the Smith strain of MCMV led to escape from NK cell control in the resistant B6 strain,29 with only a weak effect observed in the susceptible 129/J strain.29 Recent analyses have shown that the m157 molecule is a cell-surface glycophosphatidylinositol (gpi)-anchored protein that does not require b2m or transporter associated with antigen processing 1 (TAP1) for expression.30 The expression profile of m157 in the context of infection of resistant and susceptible mouse strains remains to be investigated and it is likely to provide important information about the kinetics of interaction between this molecule and its receptors. It is also worth noting that expansion of Ly49H-positive NK cells is observed during the late stages of infection in an MCMV-specific manner (i.e. not observed in other viral infections).31 The impact that the late expansion of Ly49H-positive NK cells has on the ongoing control of infection and/or the generation of adaptive antiviral immune responses remains to be evaluated. Variation in the NKC and implications for host resistance. Cmv1mediated resistance was first defined in the moderately resistant B6 mouse strain.18 Surveys of acute virus replication in the spleens of a range of inbred mouse strains indicated that resistance-like phenotypes were relatively uncommon with only the MA/My strain showing an NK cell-mediated control of viral replication that was reversed following depletion of NK cells with anti-NK1.1 monoclonal antibody (MAb).32 Analyses of the NKC regions of inbred strains of mice have revealed that conserved NKC haplotypes exist33–35; interestingly, the B6 NKC haplotype was found to be uncommon among the mouse strains tested. In addition, more recent analysis of outbred wild mice has provided independent evidence for the existence of haplotypes within the NKC region, and has confirmed that the B6-like haplotype and Cmv1-like resistance are uncommon in the outbred mouse populations studied.36 Consequently, as expected, most outbred mice in the population tested are susceptible to MCMV infection. Given the paucity of Cmv1-like alleles in outbred mouse strains, further analysis has been conducted to identify other MCMV resistance genes. Other non-H2 resistance genes – Cmv2, Cmv3 and Cmv4. The New Zealand inbred mouse strains NZW and NZB were previously found to share very similar haplotypes across the NKC region.34 Recent analyses of MCMV resistance phenotypes in these strains revealed that the NZW strain is resistant and NZB is susceptible.37 NZW resistance can be reversed by anti-NK1.1 MAb treatment, but not by anti-Ly49H (3D10) MAb treatment, despite the fact that NZW NK cells express receptors that crossreact with 3D10. Furthermore, the NZW NK cells do not bind to the viral glycoprotein m157. Altogether these findings suggest that MCMV resistance in the NZW strain is Cmv1-independent. Further analysis of the genetic control of resistance in the NZW strain indicated that a multigenic mode of resistance (collectively termed Cmv2) operates in this strain with contributions from loci that map to chromosomes 17 and X.37 The genes encoded by these loci remain to be identified. Immunology and Cell Biology


Two recent studies have analyzed the NK cell-mediated MCMV resistance that operates in the MA/My inbred strain.10,11 Resistance in this strain was found to be Ly49h-independent since MA/My NK cells do not bind the anti-Ly49H MAb or soluble m157 proteins,11 and indeed the Ly49h/Klra8 gene was found not be expressed in this strain.10 Based on genetic analyses both studies found that the resistance of NK cells in this strain was H2k dependent. In the study by Desrosier et al.,10 the Cmv3 locus was defined as being linked to the Klra gene cluster in the NKC, and the Klra16MA/My (or Ly49PMA/My) NK cell activation receptor was shown to be involved in recognition of MCMV-infected cells. As recognition of MCMV-infected cells in MA/My mice could be blocked either by anti-H2Dk or anti-Ly49P antibodies, NK cell-mediated virus resistance in this strain might involve interactions between the NK cell receptor Ly49P and the H2 molecule Dk. In addition to innate H2-mediated control of MCMV infection, Dighe et al.,11 using crosses between MA/My and C57L inbred mice, identified further contributions from both H2 and nonH2 loci in mediating resistance. Collectively, these findings support an important role for NK cells in innate H2-linked protection in MCMV infection, and suggest that NK cell receptors may be rapidly selected for specific viral sensing. In a recent study a panel of wild-derived inbred strains was screened for resistance to MCMV infection and identified the PWK/Pas strain as having a resistance phenotype similar to that of B6 mice.38 That is, resistance in PWK/Pas mice was found to be NK cell-dependent and mediated by an autosomal dominant gene that is linked to the NKC region on mouse chromosome 6. The PWK/Pas strain was also found to have a genetic profile in the Ly49H region that is similar to that seen in B6 mice. However, PWK/Pas mice infected with an MCMV mutant lacking the m157 viral glycoprotein remained resistant to MCMV infection. Furthermore, PWK/Pas NK cells failed to lyse m157-expressing target cells.38 These data indicate that MCMV resistance in the PWK/ Pas mouse strain is mediated by a novel resistance locus, named Cmv4, which most likely encodes a yet to be defined activating NK cell receptor.38 Investigations of whether MCMV-encoded class I-like molecules other than m157 serve as ligands for the PWK/Pas NK cell activating receptor will provide useful insights as to how common this mechanism of NK cell recognition of virus-infected cells is. The recent analyses summarized above have provided important evidence that different mouse strains have evolved a range of strategies for NK cell-mediated control of MCMV infection to limit the severity of acute viral infection. An implication of these studies is that a spectrum of similar effector mechanisms is likely to exist in humans as a means to combat HCMV infections. MCMV host resistance genes identified by forward genetics Classical genetics approaches based on systematic mapping and positional cloning of resistance loci that control defined phenotypic traits in inbred strains of mice have provided the basis for many resistance gene identification studies. However, the forward genetics approach has recently been adopted as a powerful alternative strategy to identify host resistance genes (reviewed by Beutler et al.39). This approach is based on the generation of random germline mutants through use of the mutagen N-ethyl-N-nitrosourea (ENU). The forward genetics approach can be used to generate libraries of point mutants that represent a ‘resistome’ to particular infectious agents. For instance, an ENU-induced mutant carrying a mutation in the Tlr9 gene was used to demonstrate that the Toll-like receptor (TLR) TLR9, which signals through the MyD88 adaptor molecule, plays an important role in regulating the level of MCMV infectioninduced IFN-a/b and subsequent NK cell activation.40 A more recent Immunology and Cell Biology

MCMV infection screen of an ENU mutant library has identified eight independent mutants that show differing disease outcomes following MCMV infection.41 One of these mutants, called Domino, was identified as having a point mutation in the DNA binding domain of signal transducer and activator of transcription 1 (STAT1), which is involved in signal transduction via the Type I IFN receptor (IFNAR). These studies illustrate the utility of the forward genetics approach for the identification of key host resistance mechanisms. Despite the utility of this approach, forward genetics will not identify differences owing to allelism at the level of gene sequence or at the level of gene expression. Hence, both classical positional cloning and forward genetics approaches should be considered for defining the contribution of host resistance genes to susceptibility and/or resistance to MCMV infection. EFFECTS OF HOST GENOTYPE ON THE INTERPLAY BETWEEN IMMUNE EFFECTORS Over the last few years, it has become increasingly clear that effective immune responses require interactions between different components of the immune system and the notion that innate and adaptive immunity are independent and standalone has become clearly redundant. The murine model of CMV infection has proven ideal to dissect the complexity of antiviral immunity, and studies conducted over the last 5 years have provided important evidence of novel interactions and interplays that occur between components of the immune system and their contribution to the control of viral infection. Importantly, the availability of the well-characterized mouse models of MCMV resistance and susceptibility have also permitted the analysis of the effects of host genotype on the interplays that take place between immune effectors during the course of MCMV infection and their contribution to viral pathogenesis and disease outcome. As discussed above, NK cells play a critical role in the early control of MCMV infection and NK cell activation induced by ligation of the Ly49H receptor allows B6 mice to effectively control viral replication in the spleen and lung. Our analysis of the effectors involved in the immune response to MCMV infection also identified dendritic cells (DCs) as being important. DCs are the antigen presenting cells par excellence, critical in the initiation and regulation of antigen-specific immunity.42 More recently, it has become clear that DCs can also regulate several aspects of innate immunity.43 In MCMV infection DCs are also a principal target of the virus.44 Importantly, infection of DCs results in early activation followed by impairment of DC function. Early after MCMV infection, DCs become activated and contribute to the generation of both innate and adaptive immune responses.44,45 This phase is followed by viral-induced impairment of DC function.44,46 The early activation of DCs induced by MCMV leads to the production of the inflammatory cytokines IFN-a, IL-12 and IL-18, which contribute to the activation of NK cell responses.45 Indeed, adoptive transfer of DCs activated ex vivo by exposure to MCMV leads to improved control of infection in vivo through the activation of NK cells.45 Interestingly, we also noted further interactions between DCs and NK cells, which vary depending on host genotype. Thus, in MCMV-susceptible BALB/c mice we noted the early disappearance of CD8a+DCs,47 one of the main DC subsets described in the mouse.48 In contrast, MCMV-resistant B6 mice retained the CD8a+DC population during MCMV infection.47 CD8a+DCs have been reported to play a critical role in the generation of virus-specific T-cell responses in a number of viral infection models through their capacity to crosspresent viral antigens.49 Although in MCMV infection, the requirement for cross-presentation remains unclear, because the virus can directly infect DCs, the significance of the differences observed in the


fate of CD8a+DCs in MCMV-resistant and susceptible mouse strains remains to be defined. What is clearer is the impact of CD8a+DCs on NK cell responses. Thus, in MCMV-resistant B6 mice, CD8a+DCs are required for the proliferation of the Ly49H+ NK cells that characterizes the late stage of acute infection.47 Whereas the impact of Ly49H+ NK cell proliferation in relation to the generation and/or maintenance of adaptive antiviral immunity is not clear, the current findings demonstrate that activation of NK cells requires input from DCs. Importantly, the available data suggest that host genotype influences the type of interactions that occur between immune effectors, an issue that needs careful consideration in follow up studies. As we will discuss in the following sections, MCMV possesses an armory of viral genes that confer upon the virus a survival advantage. Therefore, it is worth taking into account the possibility that the interactions taking place between immune effectors during MCMV infection may be influenced directly by the virus as an attempt to improve long-term survival and future studies should be designed to address this possibility. CONTRIBUTIONS OF DIVERSITY IN VIRAL GENES TO ENHANCED VIRAL SURVIVAL HCMV has been shown to exhibit extensive genetic variability in a range of genes that code for proteins with diverse functions, including structural envelope glycoproteins, regulatory proteins such as the major immediate early protein (ppUL123), and proteins that contribute to immune evasion. This has recently been comprehensively reviewed by Pignatelli et al.50 It is worth noting that although differences between laboratory strains and clinical isolates are an important issue in HCMV research, differences between viral strains are seldom considered in MCMV research, despite the fact that at least two laboratory strains of virus (Smith and K181) are utilized. Furthermore, with the advent of bacterial artificial chromosome (BAC) mutagenesis, a number of BACs derived from Smith and K181 have been constructed as backbones for further mutagenesis of specific viral genes.51,52 The effects of viral passage in tissue culture and/or in vivo on viral virulence have been clearly established.53,54 The role of cell source, time of harvest and host genotype has also been partly investigated.54 A comprehensive analysis of infectivity and virulence, of at least the two commonly used MCMV strains, after passage in an extended panel of cell types in vitro or in multiple host strains in vivo, remains to be undertaken. The relevance of this type of analysis is brought to notice by recent studies of the role of TLRs in the activation of antiviral immunity. Increased MCMV replication was reported in the spleens of TLR9deficient mice by some,40,55,56 but not all studies. In our studies,45 where we did not observe an effect of TLR9 in MCMV pathogenesis in the spleen, the K181 strain of MCMV was used, and the mice were infected with 104 plaque-forming units (PFU) of virus per mouse. In other studies, where TLR9 was reported to affect MCMV pathogenesis, mice were infected with the Smith strain of MCMV at doses in excess of 5104 PFU. These differences could be due to different viral strains being used, or could be accounted for by the fact that different viral doses, and therefore different numbers of viral particles, were used for infection. The principal role of TLRs is the recognition of foreign particles; it is therefore possible that the increased number of viral units administered when using MCMV-Smith (generally 4105), preferentially activates TLR9 pathways. Alternatively, different viral strains might have different access to intracellular compartments expressing TLR9. As discussed above, these studies highlight the importance of giving careful consideration to variations that may exist between viral strains, with the scope of ultimately identifying the viral gene products involved.

In this review, we will primarily focus on genetic variation in viral genes that target cellular responses of both the innate and adaptive host immune response, and how this is likely to impact the ability of the virus to evade the host immune responses and establish persistent infection. We will also review genetic diversity in viral genes that are targets for antiviral drugs. Viral genes that code for proteins that interact with NK cell receptors or their ligands NK cell-selected mutation in m157. Ly49H+ NK cells have been shown to play a major role in limiting early MCMV replication in B6 mice after activation through recognition of the m157 viral glycoprotein by the Ly49H activating receptor.27,28 The finding that NK cells specifically recognize a virally encoded glycoprotein led to the question of whether Ly49H+ NK cells could exert sufficiently strong selective pressure on the virus to lead to the emergence of viral mutants, capable of escaping Ly49H+NK cell-mediated immune surveillance. Two approaches were used to address this issue. In the first approach the K181 MCMV strain was used to sequentially infect Cmv1r/Ly49H+ mice six times.57 After these sequential infections a number of viral stocks were purified by limiting dilution. Sequence analyses of the m157 gene of viral isolates derived from sequentially passaging the virus in vivo revealed the presence of mutations in the m157 open reading frame resulting in premature stop codons or the loss of several amino-acid residues.57 These mutant m157 molecules were subsequently found not to bind to Ly49H, and the mutant viruses replicated in the spleens of resistant Cmv1r/Ly49H+ mice to the levels equivalent to those of wild-type parental virus in susceptible Cmv1s/Ly49H mice. In subsequent studies, the Smith MCMV strain was used to infect B6 severe combined immunodeficiency disease (SCID) mice.58,59 These mice lacked T and B cells, but had a fully competent NK cell repertoire. At 28 days p.i. B6 SCID mice showed raised viral titers in the spleens. Analysis of viral isolates purified from these mice at this late stage of infection revealed a spectrum of mutations in the m157 gene resulting in premature stop codons or loss/gain of codons coding for specific amino-acid residues. These viruses were also found to be functionally defective in binding to the Ly49H receptor and showed enhanced pathogenicity in vivo in B6 mice. Together, the studies in immunocompetent B6 and immunodeficient B6 SCID mice have revealed that Ly49H+ NK cells can provide sufficient selective pressure to rapidly and specifically drive the emergence of immune escape mutants in MCMV m157. Natural variation in m157. Previous analysis of m157–Ly49 interactions showed that the Smith strain m157 molecule could bind to the Ly49H NK cell activation receptor from B6 mice and to the NK cell inhibitory Ly49I molecule from 129/J mice.27 The MCMV m157 gene in wild-derived isolates showed considerable natural variation, with sequence variants falling into three groups based on the levels of amino-acid identity with the Smith and K181 laboratory strains.57 These groups were either 100% identical or shared identity levels of 80.4–87.9% and 63.9–68.2%, respectively. Apart from the two strains that shared 100% identity (K4 and K17A), only the G1F and N5 m157 molecules, which have the closest identity to the Smith sequence (86.7 and 84.6% identity, respectively) could bind to Ly49H.57 Our recent analyses (Corbett et al., unpublished data) indicate that the m157 sequence variants show distinct patterns of binding to NK cells from different inbred strains. These findings suggest that MCMV has evolved diversity in its m157 sequence to provide it with the ability to bind to NK cell receptors other than the Ly49H activation receptor, Immunology and Cell Biology


and that m157 may primarily act as an immunoevasin by interacting and interfering with the functions of NK cells. The concept of host resistance to CMV infection is context dependent. Resistance to MCMV in the B6 mouse strain is governed, at least in part, by the Ly49H-mediated mechanism, which is dependent on specific recognition of infected cells that express the virally encoded m157 molecule. However, infection of the normally MCMVresistant B6 mice by m157 variants that do not bind Ly49H results in high levels of viral replication and increased susceptibility to infection caused by these viral isolates is observed in this mouse strain.57 Variation in other MCMV genes affecting NK cell recognition. Other MCMV genes that affect NK cell function include the m144 gene, which encodes a MHC class I homolog that is capable of binding b2-microglobulin, but not endogenous peptides.60,61 Disruption of the expression the m144 gene attenuated viral growth in vivo and this could be reversed by depletion of NK cells.61 The MCMV m145, m152 and m155 genes encode viral proteins that downmodulate expression of the cellular MULT-1, H60 and RAE-1 stress-related molecules, respectively; these molecules are specifically recognized by the NKG2D NK cell activation receptor.62–66 A recent analysis of variation in the protein sequence of these viral genes (m144, m145, m152 and m155) from wild MCMV isolates indicates that these genes are relatively highly conserved (generally greater than 95% amino-acid identity compared with laboratory strain sequences).67 It is feasible that the high degree of conservation observed may reflect the binding of these molecules to relatively nonpolymorphic host proteins (i.e. MULT-1, H60 and RAE-1) and therefore, the need to evolve is lost. Variation in the HCMV UL18 MHC class I homolog that binds the inhibitory receptor LIR-1. Previous studies have shown that the HCMV UL18 gene encodes a MHC class I-like homolog that binds to b2m and endogenous peptides.68–70 Whereas investigations of the role of UL18 in inhibiting NK cell function have resulted in contradictory outcomes,71–74 it has been definitively shown that UL18 specifically binds to the host cell surface receptor LIR-1 (also known as ILT2/CD85j), an Ig-like receptor expressed by monocytes, T cells, B cells, DCs and NK cells.75 Two recent studies have addressed the issue of what effect the natural variation in UL18 observed in clinical isolates of HCMV has on the interaction between UL18 and the LIR-1 receptor.76,77 These studies showed that sequence variation leads to different affinities of the UL18 molecule for the LIR-1 receptor, as shown by enzyme-linked immunosorbent assay, flow cytometry and Biacore analyses. Variant forms of UL18, expressed as soluble Ig fusion molecules, showed differential capacities to inhibit lysis of BHK-CD64 target cells by LIR-1 transfected NK cells.77 Cerboni et al.76 also found that soluble forms (UL18-Fc) of these UL18 variants differed in their ability to inhibit LIR-1-dependent protection of HLA-G1+ target cells from lysis by LIR-1+ NK cells. Together, these data indicate that sequence variability in HCMV UL18 affects the ability of UL18 to modify NK cell function. What remains to be defined is whether the UL18 sequence variation has functional consequences for HCMV disease outcome in vivo. Viral genes that code for proteins that are recognized by or that modulate the functions of cytotoxic T cells MCMV and ie1. In BALB/c mice, the cytotoxic T-lymphocyte (CTL) response to MCMV is predominantly directed to the H2 Ld-restricted nonameric peptide 168-YPHFMPTNL-176 of the pp89 protein encoded by the ie1 (m123) gene.78–80 Adoptive transfer of pp89specific CTL into naı¨ve BALB/c mice is sufficient to confer protection from challenge with lethal doses of MCMV.81 Analysis of natural Immunology and Cell Biology

sequence variation in the ie1 gene of MCMV identified extensive variation in the nonapeptide sequence that defines the immunodominant epitope.82 Importantly, these sequence variants are not efficiently recognized by CTL specific for the wild-type YPHFMPTNL sequence present in the laboratory strains Smith or K181 and indeed preimmunization with the YPHFMPTNL peptide conferred protection against K181 infection, but did not reduce the pathogenesis of MCMV isolates carrying sequence variants of this nonapeptide.82 The extent of genetic variation in the pp89-encoded nonapeptide from wild-derived MCMV isolates might suggest that these variants have arisen from immune-driven pressure that has selected for ie1 escape mutants. However, we have found that repeated sequential passage of the K181 strain through H2Ld BALB/c mice does not select for mutations in the ie1 gene (Scalzo et al., unpublished observations). This finding suggests that while this protein elicits a host protective CTL response, unlike the rapid Ly49H+ NK cell driven evolution of m157 in B6 mice,57–59 the CTL pressure in BALB/c mice is insufficient to drive the evolution of the ie1 epitope. Alternative explanations are that this region of the pp89 phosphoprotein is functionally conserved or that the wild-type K181 strain sequence in maintained to skew the CTL response in mice with the H2d haplotype. The latter possibility warrants further investigation. HCMV CTL epitopes. The specificity of CTLs specific for HCMV is largely directed towards the HCMV tegument phosphoprotein pp65.83,84 A survey of multiple HLA-A*0201-restricted CTL epitopes in the pp65 protein indicated that they were conserved among different strains of HCMV.85 The UL123-encoded 72 kDa IE1 immediate early protein also represents an important HCMV CTL epitope86,87 and, while natural sequence variation exists in the IE1 epitope (IE1199–206) of clinical isolates, T-lymphocyte responses are able to crossreact with these sequence variants.88 Variation in CMV genes that code for immunoevasins that target MHC class I presentation. In a preliminary analysis of the four HCMV genes (US2, US3, US6 and US11) that regulate the cell surface expression of human MHC class I, and thus antigen presentation to CD8+ T cells, we found that the levels of sequence variation among six clinical isolates and two laboratory strains (AD169 and Towne) were relatively low with the minimum level of amino-acid identity being 96% for US6 (Scalzo and Allan, unpublished data). These data suggest that these genes may be conserved because they bind to relatively structurally conserved regions of MHC class I molecules and hence are not the subject of significant evolutionary pressure. MCMV encodes three immunoevasins that target the MHC class I presentation pathway and inhibit CTL activation. The m06-encoded gp48 and m152-encoded gp40 glycoproteins downregulate surface expression of MHC class I molecules by redirecting them to lysosomal compartments (gp48)89 or by retaining them in the ER/golgi (gp40).90,91 In contrast, the m04-encoded gp34 glycoprotein binds MHC class I molecules in the ER and on the cell surface,92,93 and inhibits CTL activity without blocking MHC class I surface expression.92 The ability of MCMV Smith m04, m06 and m152 to block MHC class I presentation is haplotype dependent94,95 and the effect on CTL recognition varies depending on the CTL epitope tested.96 As MHC haplotypes vary between mouse strains or individual wild mice, it is conceivable that variation in these immunoevasins would confer selective advantages in different host populations. Sequence variation in these genes between wild mouse-derived MCMV isolates has recently been addressed. Both m06 and m152 appear to be relatively


conserved between strains tested, whereas remarkable sequence diversity exists for m04, with m04 sequences forming six distinct groups (Smith et al.,67 Corbett et al., unpublished data). The functional implications of this variation are yet to be investigated, but the data suggest selection by different host haplotypes. It has been suggested that the lack of sequence variation in m152 may be due to functional constraints since, in addition to interfering with class I molecules, m152/gp40 also acts to downregulate the RAE-1 family of NKG2D ligands,67 as discussed above. Alternatively, these glycoproteins may bind to relatively structurally conserved regions of MHC class I molecules, and hence may not be the subject of significant evolutionary pressure. The gp34 protein contains a CTL epitope recognized by protective CTLs,97 which is conserved between wild MCMV strains (Corbett et al., unpublished data). The m04, m06 and m152 gene products have both cooperative and antagonistic effects on inhibition of MHC class I surface expression.94,95 Recently, m04 has been suggested to be both a positive and negative regulator of CTL activation98 and the combined action of all three genes may in fact misdirect, rather than block the host–CTL response. Understanding how the sequence variation between different MCMV strains relates to the host MHC haplotype and the individual CTL epitopes presented may be the key to unraveling the apparent contradiction between the MHC class I downregulation induced by these MCMV genes and the active and effective CTL response mounted by the host.99 Genetic variation in viral genes that are targets of antiviral drugs Of the eleven loci necessary for transient complementation of OriLytdependent DNA replication, CMV replication is dependent upon seven key genes involved in reproducing the double-stranded DNA.100–102 These include the CMV DNA polymerase catalytic subunit (HCMV pUL54, MCMV pM54), the DNA polymerase accessory protein (pUL44, pM44), the single-stranded DNA-binding protein (pUL57, pM57), the primase (pUL70, pM70), the helicase (pUL105, pM105), the helicase–primase associated factor (pUL102, pM102) and the OriLyt initiator protein (pUL84).100–102 Genes involved in CMV DNA replication are typically highly invariant in wild-type CMV strains (Scott, unpublished observations). Most currently available anti-CMV agents inhibit CMV DNA replication via different mechanisms of interaction with the CMV DNA polymerase catalytic subunit (pUL54).103 The HCMV-encoded protein kinase (pUL97) is also important for activation and phosphorylation of the most commonly used of these antiviral agents – the nucleoside analog, ganciclovir (GCV).104,105 The DNA polymerase and protein kinase genes are highly conserved in antiviral sensitive (naı¨ve) strains,106–108 and any significant genetic variation in these genes is predominantly the result of antiviral selective pressure.109–112 These patterns of DNA polymerase and protein kinase variation are similar to those seen in the MCMV homologs of these genes (M54 and M97, respectively), both for antiviral-sensitive strains and in vitro generated resistant isolates.113 Therefore, antiviral pressure drives mutation within the genes of replication in an analogous way as host immune pressure drives mutations within other genes. Removal of antiviral pressure either in vivo or in vitro, can lead to the reemergence of wild-type DNA polymerase and protein kinase sequence,114 although emerging evidence suggests certain antiviral resistant genotypes can persist in some individuals in the absence of antiviral pressure.112 The emergence of resistant CMV is also related to the degree of host immune competence; CMV challenge results in increased rates of viral replication in CMV-seronegative versus CMV-seropositive

immunosuppressed transplant recipients115 and good evidence exists that rates of CMV-resistant genotypes are higher in more highly immunosuppressed patients such as bone marrow recipients.116 Kinetic studies also suggest antiviral-resistant strains emerge alongside increased rates of wild-type viral replication in patients receiving subtherapeutic doses of antiviral drugs.114 This suggests host immunosuppression leads to the loss of control of viral replication with the capacity for mutants to emerge increased in such patients.114,115 The antiviral-resistant mutations generally arise within functional domains of the CMV DNA polymerase and protein kinase, interfering with processes such as substrate binding and incorporation, DNA template interaction or phosphotransfer.104,105,117,118 However, these changes are such that normal biological functions of the produced proteins is maintained and viral replication can still occur, albeit at reduced levels for certain mutations.119–121 The significant difference between mutations of DNA polymerase and protein kinase that confer resistance to antiviral drugs, and those occurring in CMV genes under immune pressure, is that mutations will be focused in certain areas because of the functional constraints of these essential replication genes.101,122 Consistent with this is the finding that certain mutations can confer resistance to different drugs of completely different inhibitory mechanisms; for example, a DNA polymerase mutation arising under GCV selective pressure can confer cross-resistance to different classes of nucleoside analogs (cidofovir) as well as the pyrophosphate analog, foscarnet.121 Interestingly, similar mutations are seen in the MCMV genome under selective pressure from the same antivirals as seen in human CMV,113 again consistent with the pressure resulting from functional constraints, rather than hostderived constraints. Conclusions The outcome of the interaction between infectious agents and their hosts is dependent on a variety of intrinsic factors pertaining to the pathogen and host. In the case of CMV the outcome of infection of immunocompetent hosts is usually asymptomatic infection and the establishment of persistence, but disease is pronounced in immunodeficient individuals. Analyses of host resistance genes controlling MCMV infection in mice have revealed a number of strategies that are mouse-strain dependent and, based on forward genetics approaches, likely to be conserved among individual animals in the population. The outcomes of host infection are highly dependent on the balance of host and viral factors that interact to shape infection and thus conclusions based on the use of certain combinations of host and viral strains may provide a limited perspective of the spectrum of biological outcomes. ACKNOWLEDGEMENTS We acknowledge financial support from the NH&MRC, ARC, The Wellcome Trust and Roche Pharmaceuticals. AAS and MADE are supported by fellowships from the NH&MRC. AJC is the recipient of a WA & MG Saw Medical Research Fellowship from the University of Western Australia. We also acknowledge Michael Brown for comments on the manuscript.

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