Recombinant Murine Gamma Herpesvirus 68 Carrying KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice

Human gamma herpesviruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are capable of inducing tumors, particularly in in immune-compromised individuals. Due to the stringent host tropism, rodents are resistant to infection by human gamma herpesviruses, creating a significant barrier for the in vivo study of viral genes that contribute to tumorigenesis. The closely-related murine gamma herpesvirus 68 (γHV68) efficiently infects laboratory mouse strains and establishes robust persistent infection without causing apparent disease. Here, we report that a recombinant γHV68 carrying the KSHV G protein-coupled receptor (kGPCR) in place of its murine counterpart induces angiogenic tumors in infected mice. Although viral GPCRs are conserved in all gamma herpesviruses, kGPCR potently activated downstream signaling and induced tumor formation in nude mouse, whereas γHV68 GPCR failed to do so. Recombinant γHV68 carrying kGPCR demonstrated more robust lytic replication ex vivo than wild-type γHV68, although both viruses underwent similar acute and latent infection in vivo. Infection of immunosuppressed mice with γHV68 carrying kGPCR, but not wild-type γHV68, induced tumors in mice that exhibited angiogenic and inflammatory features shared with human Kaposi’s sarcoma. Immunohistochemistry staining identified abundant latently-infected cells and a small number of cells supporting lytic replication in tumor tissue. Thus, mouse infection with a recombinant γHV68 carrying kGPCR provides a useful small animal model for tumorigenesis induced by a human gamma herpesvirus gene in the setting of a natural course of infection.     

Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gamma HV-68; also referred to as MHV-68) is a gammaherpesvirus which infects murid rodents. Previous studies showed that CD8 T cells are important for controlling gamma HV-68 replication during the first 2 weeks of infection and suggested a role for B cells in latent or persistent gamma HV-68 infection. To further define the importance of B cells and CD8 T cells during acute and chronic gamma HV-68 infection, we examined splenic infection in mice with null mutations in the transmembrane domain of the mu-heavy-chain constant region (MuMT; B-cell and antibody deficient) or in the beta2-microglobulin gene (beta2 -/-; CD8 deficient). Immunocompetent mice infected intraperitoneally with gamma HV-68 demonstrated peak splenic titers 9 to 10 days postinfection, cleared infectious virus 15 to 20 days postinfection, and harbored low levels of latent virus at 6 weeks postinfection. Beta2-/- mice showed peak splenic gamma HV-68 titers similar to those of normal mice but were unable to clear infectious virus completely from the spleen, demonstrating persistent infectious virus 6 weeks postinfection. These data indicate that CD8 T cells are important for clearing infectious gamma HV-68 from the spleen. Infected MuMT mice did not demonstrate detectable infectious gamma HV-68 in the spleen at any time after infection, indicating that mature B lymphocytes are necessary for acute splenic infection by gamma HV-68. Despite the lack of measurable acute infection, MuMT spleen cells harbored latent virus 6 weeks postinfection at a level about 100-fold higher than that in normal mice. These data demonstrate establishment of latency by a herpesvirus in an organ in the absence of acute viral replication in that organ. In addition, they demonstrate that gamma HV-68 can establish latency in a cell type other than mature B lymphocytes.     

Disruption of the murine gammaherpesvirus 68 M1 open reading frame leads to enhanced reactivation from latency

Murine gammaherpesvirus 68 (gammaHV68, or MHV-68) is a genetically tractable, small animal model for the analysis of gammaherpesvirus pathogenesis. The gammaHV68 genome is colinear with the genomes of other sequence gammaherpesviruses, containing large blocks of conserved genes interspersed by a number of putative genes without clear homologs in the other gammaherpesviruses. One of these putative unique genes, the M1 open reading frame (ORF), exhibits sequence homology to a poxvirus serine protease inhibitor, SPI-1, as well as to another gammaHV68 gene, M3, which we have recently shown encodes an abundantly secreted chemokine binding protein. To assess the contribution of the M1 ORF to gammaHV68 pathogenesis, we have generated a recombinant gammaHV68 in which the M1 ORF has been disrupted through targeted insertion of a lacZ expression cassette (M1.LacZ). Although M1.LacZ replicated normally in tissue culture, it exhibited decreased splenic titers at days 4 and 9 postinfection in both immunocompetent and immunodeficient mice. Despite decreased levels of acute virus replication, M1.LacZ established a latent infection comparable to wild-type (wt) gammaHV68, but exhibited an approximately fivefold increase in efficiency of reactivation from latency. M1.LacZ also caused severe vasculitis of the great elastic arteries in gamma interferon receptor (IFN-gammaR)-deficient mice with a frequency comparable to wt gammaHV68, but did not cause the mortality or splenic pathology observed with wt gammaHV68 infection of IFN-gammaR-deficient mice. Restoration of M1 ORF sequences into M1.LacZ (M1 marker rescue, or M1.MR) demonstrated that M1.LacZ phenotypic alterations in growth in vivo and latency were not due to the presence of additional mutations located elsewhere in the M1. LacZ genome. Generation of a second M1 mutant virus containing a deletion at the 5' end of the M1 ORF (M1Delta511), but lacking the LacZ expression cassette, revealed the same latency phenotype observed with the M1.LacZ mutant. However, M1Delta511 was not attenuated for acute virus replication in the spleen. We conclude that (i) the induction of arteritis in gammaHV68-infected IFN-gammaR-deficient mice can occur in the absence of splenic pathology and mortality, (ii) replication during acute infection is not the primary determinant for the establishment of latent infection, and (iii) the M1 ORF, or a closely linked gene, encodes a gene product that functions to suppress virus reactivation.     

An optimized CD4 T-cell response can control productive and latent gammaherpesvirus infection

CD4 T cells are important for control of infection with murine gammaherpesvirus 68 (gamma HV68), but it is not known whether CD4 T cells function via provision of help to other lymphocyte subsets, such as B cells and CD8 T cells, or have an independent antiviral function. Moreover, under conditions of natural infection, the CD4 T-cell response is not sufficient to eliminate infection. To determine the functional capacities of CD4 T cells under optimal or near-optimal conditions and to determine whether CD4 T cells can control gamma HV68 infection in the absence of CD8 T cells or B cells, we studied the effect of ovalbumin (OVA)-specific CD4 T cells on infection with a recombinant gamma HV68 that expresses OVA. OVA-specific CD4 T cells limited acute gamma HV68 replication and prolonged the life of infected T-cell receptor-transgenic RAG (DO.11.10/RAG) mice, demonstrating CD4 T-cell antiviral activity, independent of CD8 T cells and B cells. Despite CD4 T-cell-mediated control of acute infection, latent infection was established in DO.11.10/RAG mice. However, OVA-specific CD4 T cells reduced the frequency of latently infected cells both early (16 days postinfection) and late (42 days postinfection) after infection of mice containing CD8 T cells and B cells (DO.11.10 mice). These results show that OVA-specific CD4 T cells have B-cell and CD8 T-cell-independent antiviral functions in the control of acute infection and can, in the absence of preexisting CD8 T-cell or B-cell immunity, inhibit the establishment of gammaherpesvirus latency.     

A replication-defective gammaherpesvirus efficiently establishes long-term latency in macrophages but not in B cells in vivo

Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.     

Alpha/beta interferons regulate murine gammaherpesvirus latent gene expression and reactivation from latency

Alpha/beta interferon (IFN-alpha/beta) protects the host from virus infection by inhibition of lytic virus replication in infected cells and modulation of the antiviral cell-mediated immune response. To determine whether IFN-alpha/beta also modulates the virus-host interaction during latent virus infection, we infected mice lacking the IFN-alpha/beta receptor (IFN-alpha/betaR(-/-)) and wild-type (wt; 129S2/SvPas) mice with murine gammaherpesvirus 68 (gammaHV68), a lymphotropic gamma-2-herpesvirus that establishes latent infection in B cells, macrophages, and dendritic cells. IFN-alpha/betaR(-/-) mice cleared low-dose intranasal gammaHV68 infection with wt kinetics and harbored essentially wt frequencies of latently infected cells in both peritoneum and spleen by 28 days postinfection. However, latent virus in peritoneal cells and splenocytes from IFN-alpha/betaR(-/-) mice reactivated ex vivo with >40-fold- and 5-fold-enhanced efficiency, respectively, compared to wt cells. Depletion of IFN-alpha/beta from wt mice during viral latency also significantly increased viral reactivation, demonstrating an antiviral function of IFN-alpha/beta during latency. Viral reactivation efficiency was temporally regulated in both wt and IFN-alpha/betaR(-/-) mice. The mechanism of IFN-alpha/betaR action was distinct from that of IFN-gammaR, since IFN-alpha/betaR(-/-) mice did not display persistent virus replication in vivo. Analysis of viral latent gene expression in vivo demonstrated specific upregulation of the latency-associated gene M2, which is required for efficient reactivation from latency, in IFN-alpha/betaR(-/-) splenocytes. These data demonstrate that an IFN-alpha/beta-induced pathway regulates gammaHV68 gene expression patterns during latent viral infection in vivo and that IFN-alpha/beta plays a critical role in inhibiting viral reactivation during latency.     

Identification and sequencing of a novel rodent gammaherpesvirus that establishes acute and latent infection in laboratory mice

Gammaherpesviruses encode numerous immunomodulatory molecules that contribute to their ability to evade the host immune response and establish persistent, lifelong infections. As the human gammaherpesviruses are strictly species specific, small animal models of gammaherpesvirus infection, such as murine gammaherpesvirus 68 (γHV68) infection, are important for studying the roles of gammaherpesvirus immune evasion genes in in vivo infection and pathogenesis. We report here the genome sequence and characterization of a novel rodent gammaherpesvirus, designated rodent herpesvirus Peru (RHVP), that shares conserved genes and genome organization with γHV68 and the primate gammaherpesviruses but is phylogenetically distinct from γHV68. RHVP establishes acute and latent infection in laboratory mice. Additionally, RHVP contains multiple open reading frames (ORFs) not present in γHV68 that have sequence similarity to primate gammaherpesvirus immunomodulatory genes or cellular genes. These include ORFs with similarity to major histocompatibility complex class I (MHC-I), C-type lectins, and the mouse mammary tumor virus and herpesvirus saimiri superantigens. As these ORFs may function as immunomodulatory or virulence factors, RHVP presents new opportunities for the study of mechanisms of immune evasion by gammaherpesviruses.     

A Gammaherpesvirus Establishes Persistent Infection in Neuroblastoma Cells

Gammaherpesvirus (γHV) infection of the central nervous system (CNS) has been implicated in diverse neurological diseases, and murine γHV-68 (MHV-68) is known to persist in the brain after cerebral infection. The underlying molecular mechanisms of persistency of virus in the brain are poorly understood. Here, we characterized a unique pattern of MHV-68 persistent infection in neuroblastoma cells. On infection with MHV-68, both murine and human neuroblastoma cells expressed viral lytic proteins and produced virions. However, the infected cells survived productive infection and could be cultured for multiple passages without affecting their cellular growth. Latent infection as well as productive replication was established in these prolonged cultures, and lytic replication was further increased by treatment with lytic inducers. Our results provide a novel system to study persistent infection of γHVs in vitro following de novo infection and suggest application of MHV-68 as a potential gene transfer vector to the brain.¹     

Murine gammaherpesvirus 68 open reading frame 75c tegument protein induces the degradation of PML and is essential for production of infectious virus

Promyelocytic Leukemia nuclear body (PML NB) proteins mediate an intrinsic cellular host defense response against virus infections. Herpesviruses express proteins that modulate PML or PML-associated proteins by a variety of strategies, including degradation of PML or relocalization of PML NB proteins. The consequences of PML-herpesvirus interactions during infection in vivo have yet to be investigated in detail, largely because of the species-specific tropism of many human herpesviruses. Murine gammaherpesvirus 68 (γHV68) is emerging as a suitable model to study basic biological questions of virus-host interactions because it naturally infects mice. Therefore, we sought to determine whether γHV68 targets PML NBs as part of its natural life cycle. We found that γHV68 induces PML degradation through a proteasome-dependent mechanism and that loss of PML results in more robust virus replication in mouse fibroblasts. Surprisingly, γHV68-mediated PML degradation was mediated by the virion tegument protein ORF75c, which shares homology with the cellular formylglycinamide ribotide amidotransferase enzyme. In addition, we show that ORF75c is essential for production of infectious virus. ORF75 homologs are conserved in all rhadinoviruses but so far have no assigned functions. Our studies shed light on a potential role for this unusual protein in rhadinovirus biology and suggest that γHV68 will be a useful model for investigation of PML-herpesvirus interactions in vivo.     

Murine gammaherpesvirus 68 genes both induce and suppress lymphoproliferative disease

Gammaherpesvirus infection is associated with an increased incidence of lymphoproliferative disease in immunocompromised hosts. Murine gammaherpesvirus 68 (γHV68) infection of BALB β₂-microglobulin-deficient (BALB β₂m^−/−) mice provides an animal model for analysis of the mechanisms responsible for the induction of a lymphoproliferative disease, atypical lymphoid hyperplasia (ALH), that is pathologically similar to posttransplant lymphoproliferative disease associated with Epstein-Barr virus infection. Here we report that the γHV68 v-cyclin and v-bcl-2 genes are required for the efficient induction of γHV68-associated ALH in BALB β₂m^−/− mice, while the v-GPCR gene is dispensable for ALH induction. In contrast to these findings, deletion of the viral M1 gene enhanced ALH. Thus, γHV68 genes can either inhibit or enhance the induction of lymphoproliferative disease in immunocompromised mice.     

Lymphotoxin-alpha-deficient mice can clear a productive infection with murine gammaherpesvirus 68 but fail to develop splenomegaly or lymphocytosis

Respiratory challenge with murine gammaherpesvirus 68 (MHV-68) leads to an acute productive infection of the lung and a persistent latent infection in B lymphocytes, epithelia, and macrophages. The virus also induces splenomegaly and an increase in the number of activated CD8 T cells in the circulation. Lymphotoxin- alpha-deficient (LTalpha(-/-)) mice have no lymph nodes and have disrupted splenic architecture. Surprisingly, in spite of the severe defect in secondary lymphoid tissue, LTalpha(-/-) mice could clear a productive MHV-68 infection, although with delayed kinetics compared to wild-type mice, and could control latent infection. Cytotoxic T-cell activity was comparable in the lungs and spleens of LTalpha(-/-) and wild-type mice. However, splenic gamma interferon responses were substantially reduced in LTalpha(-/-) mice. Furthermore, LTalpha(-/-) mice failed to develop splenomegaly or lymphocytosis. Although germinal centers were absent, LTalpha(-/-) mice were able to class switch and showed significant virus-specific antibody titers. This work demonstrates that organized secondary lymphoid tissue is not an absolute requirement for the generation of immune responses to viral infections.     

Viral Load and a Locus on Chromosome 11 Affect the Late Clinical Disease Caused by Theiler’s Virus

Theiler's virus causes a persistent infection and a demyelinating disease of mice which is a model for multiple sclerosis. Susceptibility to viral persistence maps to several loci, including the interferon gamma locus. Inactivating the gene coding for the interferon gamma receptor makes 129/Sv mice susceptible to persistent infection and clinical disease, whereas inactivating the interferon gamma gene makes C57BL/6 mice susceptible to persistent infection but not to clinical disease. This difference in phenotype is due to the difference in genetic background. Clinical disease depends on high viral load and Tmevd5, a locus on chromosome 11. These results have consequences for the identification of viruses which might be implicated in multiple sclerosis.     

Gammaherpesvirus Latency Accentuates EAE Pathogenesis: Relevance to Epstein-Barr Virus and Multiple Sclerosis

Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS), yet EBV''s role in MS remains elusive. We utilized murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, to examine how infection by a virus like EBV could enhance CNS autoimmunity. Mice latently infected with γHV-68 developed more severe EAE including heightened paralysis and mortality. Similar to MS, γHV-68EAE mice developed lesions composed of CD4 and CD8 T cells, macrophages and loss of myelin in the brain and spinal cord. Further, T cells from the CNS of γHV-68 EAE mice were primarily Th1, producing heightened levels of IFN-γ and T-bet accompanied by IL-17 suppression, whereas a Th17 response was observed in uninfected EAE mice. Clearly, γHV-68 latency polarizes the adaptive immune response, directs a heightened CNS pathology following EAE induction reminiscent of human MS and portrays a novel mechanism by which EBV likely influences MS and other autoimmune diseases.     

The anti-interferon activity of conserved viral dUTPase ORF54 is essential for an effective MHV-68 infection

Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR-/- mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68.     

Pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for CD8 T cells.

Murine gammaherpesvirus is a natural rodent pathogen which causes a primary infection in the lungs and establishes a persistent infection in B lymphocytes. During the primary infection, large amounts of gamma interferon (IFN-gamma) are produced by spleen, mediastinal, and cervical lymph node cells. To investigate the role of IFN-gamma in control of the virus infection, mice lacking the cellular receptor for IFN-gamma (IFN-gamma R-/- mice) were infected with murine gammaherpesvirus 68 (MHV68). IFN-gamma R-/- mice showed no difference from wild-type mice in the titers of infectious virus in the lungs or in the rate of clearance of the lung infection. In the spleen, however, clear differences were observed. By 14 days postinfection, spleens from IFN-gamma R-/- mice were pale, shrunken, and fibrous. Histological examination showed that there was an early (day 10) infiltration of granulocytes followed by widespread destruction of splenic architecture (days 14 to 17). A marked decrease in the number of splenic B cells and CD4+ and CD8+ T cells occurred. These changes were accompanied by a 10- to 100-fold greater load of latently infected cells in IFN-gamma R-/- mice than in wild-type mice at 14 to 17 days postinfection, but this was reduced to the levels found in wild-type mice by 21 days postinfection. Treatment of the mice with the antiviral drug 2'-deoxyl-5-ethyl-beta-4'-thiouridine from 6 days postinfection did not prevent the occurrence of these changes. The changes were, however, completely reversed by depletion of CD8+ T cells prior to and during the primary infection. Depletion of CD4+ T cells also reversed the major pathological and virological changes, although in this case there was evidence of some histological changes. Thus, the lack of IFN-gamma receptor had profound consequences in spleens of MHV68-infected mice. The possible mechanisms involved in these changes are discussed.     

Viral Bcl-2-Mediated Evasion of Autophagy Aids Chronic Infection of γHerpesvirus 68

γ-herpesviruses (γHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine γ-herpesvirus 68 (γHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant γHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which γHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of γ-herpesviruses.     

The predominant virus antigen burden is present in macrophages in Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus (TMEV) produces a persistent central nervous system infection and chronic, inflammatory demyelinating disease in susceptible mice. TMEV antigen(s) and RNA genome have been detected in astrocytes, oligodendrocytes, and macrophages during persistence. Whether there is a predominant cell type in which TMEV persists has not been resolved. Since TMEV-induced demyelinating lesions are infiltrated with macrophages and a number of other persistent viruses show near-exclusive tropism for these phagocytic cells, we used two-color immunofluorescent staining with conventional and confocal microscopy to colocalize TMEV to cells that stain with monoclonal antibodies (MOMA-2) [unknown antigen], Mac-1 [CD11b], FA-11 [CD66], and 2F8 [scavenger receptor]) to macrophages in BeAn-infected SJL mice. A predominant virus antigen burden within macrophages infiltrating demyelinating lesions was seen. A dichotomy of cells staining for virus antigen(s) was found with infected cells containing either a large or small virus antigen load. Ninety percent of cells with a large virus antigen load were large phagocytes (20 to 50 microns) that were readily detected at low power (5x objective). Cells with smaller amounts of virus antigen(s) turned out to be either these same large phagocytic cells or much smaller cells, approximately equal to 10 microns in diameter. Forty percent of cells with a small virus antigen load were macrophages. The unidentified approximately equal to 10-microns cells that are virus antigen positive and macrophage negative in this study could still be macrophages, or they may be oligodendrocytes. The fact that virus was detected in the cytoplasm and not phagolysosomes of macrophages and the sheer mass of fluorescently stained virus proteins in some macrophages suggest that TMEV persists in these phagocytic cells by active virus replication.