The gammaherpesvirus 68 latency-associated nuclear antigen homolog is critical for the establishment of splenic latency

Open reading frame 73 (ORF 73) is conserved among the gamma-2-herpesviruses (rhadinoviruses) and, in Kaposi's sarcoma-associated herpesvirus (KSHV) and herpesvirus saimiri (HVS), has been shown to encode a latency-associated nuclear antigen (LANA). The KSHV and HVS LANAs have also been shown to be required for maintenance of the viral genome as an episome during latency. LANA binds both the viral latency-associated origin of replication and the host cell chromosome, thereby ensuring efficient partitioning of viral genomes to daughter cells during mitosis of a latently infected cell. In gammaherpesvirus 68 (gammaHV68), the role of the LANA homolog in viral infection has not been analyzed. Here we report the construction of a gammaHV68 mutant containing a translation termination codon in the LANA ORF (73.STOP). The 73.STOP mutant virus replicated normally in vitro, in both proliferating and quiescent murine fibroblasts. In addition, there was no difference between wild-type (WT) and 73.STOP virus in the kinetics of induction of lethality in mice lacking B and T cells (Rag 1(-/-)) infected with 1000 PFU of virus. However, compared to WT virus, the 73.STOP mutant exhibited delayed kinetics of replication in the lungs of immunocompetent C57BL/6 mice. In addition, the 73.STOP mutant exhibited a severe defect in the establishment of latency in the spleen of C57BL/6 mice. Increasing the inoculum of 73.STOP virus partially overcame the acute replication defected observed in the lungs at day 4 postinfection but did not ameliorate the severe defect in the establishment of splenic latency. Thus, consistent with its proposed role in replication of the latent viral episome, LANA appears to be a critical determinant in the establishment of gammaHV68 latency in the spleen post-intranasal infection.     

Expansion of effector memory TCR Vbeta4+ CD8+ T cells is associated with latent infection-mediated resistance to transplantation tolerance

Therapies that control largely T cell-dependent allograft rejection in humans also possess the undesirable effect of impairing T cell function, leaving transplant recipients susceptible to opportunistic viruses. Prime among these opportunists are the ubiquitous herpesviruses. To date, studies are lacking that address the effect of viruses that establish a true latent state on allograft tolerance or the effect of tolerance protocols on the immune control of latent viruses. By using a mixed chimerism-based tolerance-induction protocol, we found that mice undergoing latent infection with gammaHV68, a murine gamma-herpesvirus closely related to human gamma-herpesviruses such as EBV and Kaposi's sarcoma-associated herpesvirus, significantly resist tolerance to allografts. Limiting the degree of virus reactivation or innate immune response did not reconstitute chimerism in latently infected mice. However, gammaHV68-infected mice showed increased frequency of CD8+ T cell alloreactivity and, interestingly, expansion of virus-induced, alloreactive, "effector/effector memory" TCR Vbeta4+CD8+ T cells driven by the gammaHV68-M1 gene was associated with resistance to tolerance induction in studies using gammaHV68-M1 mutant virus. These results define the viral gene and immune cell types involved in latent infection-mediated resistance to allograft tolerance and underscore the influence of latent herpesviruses on allograft survival.     

Host and viral genetics of chronic infection: a mouse model of gamma-herpesvirus pathogenesis.

A general association of human and primate lymphotropic herpesviruses (gamma-herpesviruses) with the development of lymphomas, as well as other tumors, especially in immunocompromised hosts, has been well documented. The lack of relevant small animal models for human gamma-herpesviruses has impeded progress in understanding the role of these viruses in the development of chronic disease. Recent research characterizing infection of inbred strains of mice with a murine gamma-herpesvirus, gamma-herpesvirus 68 (gammaHV68), is providing insights into viral and host factors involved in the establishment and control of chronic gamma-herpesvirus infection.     

Role of B-cell proliferation in the establishment of gammaherpesvirus latency

Murine gammaherpesvirus 68 (gammaHV68) provides a tractable small animal model with which to study the mechanisms involved in the establishment and maintenance of latency by gammaherpesviruses. Similar to the human gammaherpesvirus Epstein-Barr virus (EBV), gammaHV68 establishes and maintains latency in the memory B-cell compartment following intranasal infection. Here we have sought to determine whether, like EBV infection, gammaHV68 infection in vivo is associated with B-cell proliferation during the establishment of chronic infection. We show that gammaHV68 infection leads to significant splenic B-cell proliferation as late as day 42 postinfection. Notably, gammaHV68 latency was found predominantly in the proliferating B-cell population in the spleen on both days 16 and 42 postinfection. Furthermore, virus reactivation upon ex vivo culture was heavily biased toward the proliferating B-cell population. DNA methyltransferase 1 (Dnmt1) is a critical maintenance methyltransferase which, during DNA replication, maintains the DNA methylation patterns of the cellular genome, a process that is essential for the survival of proliferating cells. To assess whether the establishment of gammaHV68 latency requires B-cell proliferation, we characterized infections of conditional Dnmt1 knockout mice by utilizing a recombinant gammaHV68 that expresses Cre-recombinase (gammaHV68-Cre). In C57BL/6 mice, the gammaHV68-Cre virus exhibited normal acute virus replication in the lungs as well as normal establishment and reactivation from latency. Furthermore, the gammaHV68-Cre virus also replicated normally during the acute phase of infection in the lungs of Dnmt1 conditional mice. However, deletion of the Dnmt1 alleles from gammaHV68-infected cells in vivo led to a severe ablation of viral latency, as assessed on both days 16 and 42 postinfection. Thus, the studies provide direct evidence that the proliferation of latently infected B cells is critical for the establishment of chronic gammaHV68 infection.     

A gammaherpesvirus 68 gene 50 null mutant establishes long-term latency in the lung but fails to vaccinate against a wild-type virus challenge

The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (gammaHV68). Introduction of a translation stop codon in gammaHV68 gene 50 (gene 50.stop gammaHV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop gammaHV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop gammaHV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type gammaHV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.     

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.     

B cells regulate murine gammaherpesvirus 68 latency

The dynamics of the establishment of, and reactivation from, gammaherpesviruses latency has not been quantitatively analyzed in the natural host. Gammaherpesvirus 68 (gammaHV68) is a murine gammaherpesvirus genetically related to primate gammaherpesviruses that establishes a latent infection in infected mice. We used limiting dilution reactivation (frequency of cells reactivating gammaHV68 in vitro) and limiting dilution PCR (frequency of cells carrying gammaHV68 genome) assays to compare gammaHV68 latency in normal (C57BL/6) and B-cell-deficient (MuMT) mice. After intraperitoneal (i.p.) inoculation, latent gammaHV68 was detected in the spleen, bone marrow, and peritoneal cells. Both B-cell-deficient and C57BL/6 mice established latent infection in peritoneal cells after either i.p. or intranasal (i.n.) inoculation. In contrast, establishment of splenic latency was less efficient in B-cell-deficient than in C57BL/6 mice after i.n. inoculation. Analysis of reactivation efficiency (reactivation frequency compared to frequency of cells carrying gammaHV68 genome) revealed that (i) regardless of route or mouse strain, splenic cells reactivated gammaHV68 less efficiently than peritoneal cells, (ii) the frequency of cells carrying gammaHV68 genome was generally comparable over the course of infection between C57BL/6 and B-cell-deficient mice, (iii) between 28 and 250 days after infection, cells from B-cell-deficient mice reactivated gammaHV68 10- to 100-fold more efficiently than cells from C57BL/6 mice, (iv) at 7 weeks postinfection, B-cell-deficient mice had more genome-positive peritoneal cells than C57BL/6 mice, and (v) mixing cells (ratio of 3 to 1) that reactivated inefficiently with cells that reactivated efficiently did not significantly decrease reactivation efficiency. Consistent with a failure to normally regulate chronic gammaHV68 infection, the majority of infected B-cell-deficient mice died between 100 and 200 days postinfection. We conclude that (i) B cells are not required for establishment of gammaHV68 latency, (ii) there are organ-specific differences in the efficiency of gammaHV68 reactivation, (iii) B cells play a crucial role in regulating reactivation of gammaHV68 from latency, and (iv) B cells are important for controlling chronic gammaHV68 infection.     

Infection of dendritic cells by a gamma2-herpesvirus induces functional modulation

The murine gamma-herpesvirus-68 (gammaHV68) establishes viral latency in dendritic cells (DCs). In the present study, we examined the specific consequences of DC infection by gammaHV68, both in vivo and in vitro. Ex vivo analysis of infected mice showed that the virus colonizes respiratory DCs very early after infection and that all subsets of splenic DCs analyzed are viral targets. We have developed and characterized an in vitro model of gammaHV68 infection of DCs. Using this model, we demonstrated that viral infection neither induces full DC maturation nor interferes with exogenous activation, which is assessed by cell surface phenotypic changes. However, whereas gammaHV68 infection alone failed to elicit cytokine secretion, IL-10 secretion of exogenously activated DCs was enhanced. Furthermore, gammaHV68-infected DCs efficiently stimulated virus-specific T cell hybridomas but failed to induce alloreactive stimulation of normal T cells. These data indicate that viral infection doesn't interfere with Ag processing and presentation but does interfere with the ability of DCs to activate T cells. The inhibition of T cell activation was partially reversed by blocking IL-10. Analysis of infected mice shows elevated levels of IL-10 expression in DCs and that lack of endogenous IL-10 is associated with decreased gammaHV68 long-term latency. Taken together, these observations indicate that gamma2-herpesvirus infection of DCs is a mechanism of viral immune evasion, partially mediated by IL-10.     

Characterization of a spontaneous 9.5-kilobase-deletion mutant of murine gammaherpesvirus 68 reveals tissue-specific genetic requirements for latency

Murine gammaherpesvirus 68 (gammaHV68 [also known as MHV-68]) establishes a latent infection in mice, providing a small-animal model with which to identify host and viral factors that regulate gammaherpesvirus latency. While gammaHV68 establishes a latent infection in multiple tissues, including splenocytes and peritoneal cells, the requirements for latent infection within these tissues are poorly defined. Here we report the characterization of a spontaneous 9.5-kb-deletion mutant of gammaHV68 that lacks the M1, M2, M3, and M4 genes and eight viral tRNA-like genes. Previously, this locus has been shown to contain the latency-associated M2, M3, and viral tRNA-like genes. Through characterization of this mutant, we found that the M1, M2, M3, M4 genes and the viral tRNA-like genes are dispensable for (i) in vitro replication and (ii) the establishment and maintenance of latency in vivo and reactivation from latency following intraperitoneal infection. In contrast, following intranasal infection with this mutant, there was a defect in splenic latency at both early and late times, a phenotype not observed in peritoneal cells. These results indicate (i) that there are different genetic requirements for the establishment of latency in different latent reservoirs and (ii) that the genetic requirements for latency depend on the route of infection. While some of these phenotypes have been observed with specific mutations in the M1 and M2 genes, other phenotypes have never been observed with the available gammaHV68 mutants. These studies highlight the importance of loss-of-function mutations in defining the genetic requirements for the establishment and maintenance of herpesvirus latency.     

The murine gammaherpesvirus 68 ORF27 gene product contributes to intercellular viral spread

Herpesviruses remain predominantly cell associated within their hosts, implying that they spread between cells by a mechanism distinct from free virion release. We previously identified the efficient release of murine gammaherpesvirus 68 (MHV-68) virions as a function of the viral gp150 protein. Here we show that the MHV-68 ORF27 gene product, gp48, contributes to the direct spread of viruses from lytically infected to uninfected cells. Monoclonal antibodies to gp48 identified it on infected cell surfaces and in virions. gp48-deficient viruses showed no obvious deficit in virion cell binding, single-cycle replication, or virion release but had reduced lytic propagation between cells. After intranasal infection of mice, ORF27-deficient viruses were impaired predominantly in lytic replication in the lungs. There was a small deficit in latency establishment, but long-term latency appeared normal. Since ORF27 has homologs in both Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, it is likely part of a conserved mechanism employed by gammaherpesviruses to disseminate lytically in their hosts.     

Maintenance of gammaherpesvirus latency requires viral cyclin in the absence of B lymphocytes

Gammaherpesviruses establish a life-long chronic infection that is tightly controlled by the host immune response. We previously demonstrated that viruses lacking the gammaherpesvirus 68 (gammaHV68) viral cyclin (v-cyclin) exhibited a severe defect in reactivation from latency and persistent replication. In this analysis of chronic infection, we demonstrate that the v-cyclin is required for gammaHV68-associated mortality in B-cell-deficient mice. Furthermore, we identify the v-cyclin as the first gene product required for maintenance of gammaherpesvirus latency in vivo in the absence of B lymphocytes. While the v-cyclin was necessary for maintenance of latency in the absence of B cells, maintenance of v-cyclin-deficient viruses was equivalent to that of wild-type gammaHV68 in the presence of B cells. These results support a model in which maintenance of chronic gammaHV68 infection requires v-cyclin-dependent reactivation and reseeding of non-B-cell latency reservoirs in the absence of B cells and raise the possibility that B cells represent a long-lived latency reservoir maintained independently of reactivation. These results highlight distinct mechanisms for the maintenance of chronic infection in immunocompetent and B-cell-deficient mice and suggest that the different latency reservoirs have distinct gene requirements for the maintenance of latency.     

Establishment and maintenance of gammaherpesvirus latency are independent of infective dose and route of infection

Gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are important human pathogens that establish long-term latent infections. Understanding of the initiation and maintenance of latent infections has important implications for the prevention and treatment of gammaherpesvirus-related diseases. Although much is known about gammaherpesvirus pathogenesis, it is unclear how the infectious dose of a virus influences its ability to establish latent infection. To examine the relationship between the infectious dose and gammaherpesvirus latency, we inoculated wild-type mice with 0.01 to 10(6) PFU of murine gammaherpesvirus 68 (gammaHV68) and quantitatively measured latency and acute-phase replication. Surprisingly, during latency, the frequencies of ex vivo reactivation were similar over a 10(7)-fold range of doses for i.p. infection and over a 10(4)-fold range of doses for intranasal infection. Further, the frequencies of cells harboring viral genome during latency did not differ substantially over similar dose ranges. Although the kinetics of acute-phase replication were delayed at small doses of virus, the peak titer did not differ significantly between mice infected with a large dose of virus and those infected with a small dose of virus. The results presented here indicate that any initiation of infection leads to substantial acute-phase replication and subsequent establishment of a maximal level of latency. Thus, infections with doses as small as 0.1 PFU of gammaHV68 result in stable levels of acute-phase replication and latent infection. These results demonstrate that the equilibrium level of establishment of gammaherpesvirus latency is independent of the infectious dose and route of infection.     

Type I interferon inhibition and dendritic cell activation during gammaherpesvirus respiratory infection

The respiratory tract is a major mucosal site for microorganism entry into the body, and type I interferon (IFN) and dendritic cells constitute a first line of defense against viral infections. We have analyzed the interaction between a model DNA virus, plasmacytoid dendritic cells, and type I IFN during lung infection of mice. Our data show that murine gammaherpesvirus 68 (gammaHV68) inhibits type I IFN secretion by dendritic cells and that plasmacytoid dendritic cells are necessary for conventional dendritic cell maturation in response to gammaHV68. Following gammaHV68 intranasal inoculation, the local and systemic IFN-alpha/beta response is below detectable levels, and plasmacytoid dendritic cells are activated and recruited into the lung with a tissue distribution that differs from that of conventional dendritic cells. Our results suggest that plasmacytoid dendritic cells and type I IFN have important but independent roles during the early response to a respiratory gammaHV68 infection. gammaHV68 infection inhibits type I IFN production by dendritic cells and is a poor inducer of IFN-alpha/beta in vivo, which may serve as an immune evasion strategy.     

Antibody to a lytic cycle viral protein decreases gammaherpesvirus latency in B-cell-deficient mice

While antiviral antibody plays a key role in resistance to acute viral infection, the contribution of antibody to the control of latent virus infection is less well understood. Gammaherpesvirus 68 (gammaHV68) infection of mice provides a model well suited to defining contributions of specific immune system components to the control of viral latency. B cells play a critical role in regulating gammaHV68 latency, but the mechanism(s) by which B cells regulate latency is not known. In the experiments reported here, we determined the effect of passively transferred antibody on established gammaHV68 latency in B-cell-deficient (B-cell(-/-)) mice. Immune antibody decreased the frequency of cells reactivating ex vivo from latency in splenocytes (>10-fold) and peritoneal cells (>100-fold) and the frequency of cells carrying latent viral genome in splenocytes (>5-fold) and peritoneal cells (>50-fold). This effect required virus-specific antibody and was observed when total and virus-specific serum antibody concentrations in recipient B-cell(-/-) mice were <8% of those in normal mice during latent infection. Passive transfer of antibody specific for the lytic cycle gammaHV68 RCA protein, but not passive transfer of antibody specific for the v-cyclin protein or the latent protein M2, decreased both the frequency of cells reactivating ex vivo from latency and the frequency of cells carrying the latent viral genome. Therefore, antibody specific for lytic cycle viral antigens can play an important role in the control of gammaherpesvirus latency in immunocompromised hosts. Based on these findings, we propose a model in which ongoing productive replication is essential for maintaining high levels of latently infected cells in immunocompromised hosts. We confirmed this model by the treatment of latently infected B-cell(-/-) mice with the antiviral drug cidofovir.     

Effective vaccination against long-term gammaherpesvirus latency

The fundamental question of whether a primed immune system is capable of preventing latent gammaherpesvirus infection remains unanswered. Recent studies showing that vaccination can reduce acute replication and short-term latency but cannot alter long-term latency further call into question the possibility of achieving sterilizing immunity against gammaherpesviruses. Using the murine gammaherpesvirus 68 (gammaHV68) system, we demonstrate that it is possible to effectively vaccinate against long-term latency. By immunizing mice with a gammaHV68 mutant virus that is deficient in its ability to reactivate from latency, we reduced latent infection of wild-type challenge virus to a level below the limit of detection. Establishment of latency was inhibited by vaccination regardless of whether mice were challenged intraperitoneally or intranasally. Passive transfer of antibody from vaccinated mice could partially reconstitute the effect, demonstrating that antibody is an important component of vaccination. These results demonstrate the potential of a memory immune response against gammaherpesviruses to alter long-term latency and suggest that limiting long-term latent infection in a clinically relevant situation is an attainable goal.     

Long-term latent murine Gammaherpesvirus 68 infection is preferentially found within the surface immunoglobulin D-negative subset of splenic B cells in vivo

Murine gammaherpesvirus 68 (gammaHV68; also known as MHV-68) can establish a latent infection in both inbred and outbred strains of mice and, as such, provides a tractable small-animal model to address mechanisms and cell types involved in the establishment and maintenance of chronic gammaherpesvirus infection. Latency can be established at multiple anatomic sites, including the spleen and peritoneum; however, the contribution of distinct cell types to the maintenance of latency within these reservoirs remains poorly characterized. B cells are the major hematopoietic cell type harboring latent gammaHV68. We have analyzed various splenic B-cell subsets at early, intermediate, and late times postinfection and determined the frequency of cells either (i) capable of spontaneously reactivating latent gammaHV68 or (ii) harboring latent viral genome. These analyses demonstrated that latency is established in a variety of cell populations but that long-term latency (6 months postinfection) in the spleen after intranasal inoculation predominantly occurs in B cells. Furthermore, at late times postinfection latent gammaHV68 is largely confined to the surface immunoglobulin D-negative subset of B cells.