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.     

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.     

Gamma interferon blocks gammaherpesvirus reactivation from latency

Establishment of latent infection and reactivation from latency are critical aspects of herpesvirus infection and pathogenesis. Interfering with either of these steps in the herpesvirus life cycle may offer a novel strategy for controlling herpesvirus infection and associated disease pathogenesis. Prior studies show that mice deficient in gamma interferon (IFN-gamma) or the IFN-gamma receptor have elevated numbers of cells reactivating from murine gammaherpesvirus 68 (gammaHV68) latency, produce infectious virus after the establishment of latency, and develop large-vessel vasculitis. Here, we demonstrate that IFN-gamma is a powerful inhibitor of reactivation of gammaHV68 from latency in tissue culture. In vivo, IFN-gamma controls viral gene expression during latency. Importantly, depletion of IFN-gamma in latently infected mice results in an increased frequency of cells reactivating virus. This demonstrates that IFN-gamma is important for immune surveillance that limits reactivation of gammaHV68 from latency.     

Ex vivo stimulation of B cells latently infected with gammaherpesvirus 68 triggers reactivation from latency

Murine gammaherpesvirus 68 (gammaHV68) infection of mice results in the establishment of a chronic infection, which is largely maintained through latent infection of B lymphocytes. Acute virus replication is almost entirely cleared by 2 weeks postinfection. Spontaneous reactivation of gammaHV68 from latently infected splenocytes upon ex vivo culture can readily be detected at the early stages of infection (e.g., day 16). However, by 6 weeks postinfection, very little spontaneous reactivation is detected upon explant into tissue culture. Here we report that stimulation of latently infected splenic B cells harvested at late times postinfection with cross-linking surface immunoglobulin (Ig), in conjunction with anti-CD40 antibody treatment, triggers virus reactivation. As expected, this treatment resulted in B-cell activation, as assessed by upregulation of CD69 on B cells, and ultimately B-cell proliferation. Since anti-Ig/anti-CD40 stimulation resulted in splenic B-cell proliferation, we assessed whether this reactivation stimulus could overcome the previously characterized defect in virus reactivation of a v-cyclin null gammaHV68 mutant. This analysis demonstrated that anti-Ig/anti-CD40 stimulation could drive reactivation of the v-cyclin null mutant virus in latently infected splenocytes, but not to the levels observed with wild-type gammaHV68. Thus, there appears to be a role for the v-cyclin in B cells following anti-Ig/anti-CD40 stimulation independent of the induction of the cell cycle. Finally, to assess signals that are not mediated through the B-cell receptor, we demonstrate that addition of lipopolysaccharide to explanted splenocyte cultures also enhanced virus reactivation. These studies complement and extend previous analyses of Epstein-Barr virus and Kaposi's sarcoma-associated virus reactivation from latently infected cell lines by investigating reactivation of gammaHV68 from latently infected primary B cells recovered from infected hosts.     

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.     

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.     

Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells

B cells have previously been identified as the major hematopoietic cell type harboring latent gammaherpesvirus 68 (gammaHV68) (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). However, we have shown that gammaHV68 efficiently establishes latency in B-cell-deficient mice (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775-6780, 1996), demonstrating that B cells are not required for gammaHV68 latency. To understand this dichotomy, we determined whether hematopoietic cell types, in addition to B cells, carry latent gammaHV68. We observed a high frequency of cells that reactivate latent gammaHV68 in peritoneal exudate cells (PECs) derived from both B-cell-deficient and normal C57BL/6 mice. PECs were composed primarily of macrophages in B-cell-deficient mice and of macrophages plus B cells in normal C57BL/6 mice. To determine which cells in PECs from C57BL/6 mice carry latent gammaHV68, we developed a limiting-dilution PCR assay to quantitate the frequency of cells carrying the gammaHV68 genome in fluorescence-activated cell sorter-purified cell populations. We also quantitated the contribution of individual cell populations to the total frequency of cells carrying latent gammaHV68. At early times after infection, the frequency of PECs that reactivated gammaHV68 correlated very closely with the frequency of PECs carrying the gammaHV68 genome, validating measurement of the frequency of viral-genome-positive cells as a measure of latency in this cell population. F4/80-positive macrophage-enriched, lymphocyte-depleted PECs harbored most of the gammaHV68 genome and efficiently reactivated gammaHV68, while CD19-positive, B-cell-enriched PECs harbored about a 10-fold lower frequency of gammaHV68 genome-positive cells. CD4-positive, T-cell-enriched PECs contained only a very low frequency of gammaHV68 genome-positive cells, consistent with previous analyses indicating that T cells are not a reservoir for gammaHV68 latency (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). Since macrophages are bone marrow derived, we determined whether elicitation of a large inflammatory response in the peritoneum would recruit additional latent cells into the peritoneum. Thioglycolate inoculation increased the total number of PECs by about 20-fold but did not affect the frequency of cells that reactivate gammaHV68, consistent with a bone marrow reservoir for latent gammaHV68. These experiments demonstrate gammaHV68 latency in two different hematopoietic cell types, F4/80-positive macrophages and CD19-positive B cells, and argue for a bone marrow reservoir for latent gammaHV68.     

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.     

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.     

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.     

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.     

Establishment and maintenance of long-term murine gammaherpesvirus 68 latency in B cells in the absence of CD40

Murine gammaherpesvirus 68 (gammaHV68), like Epstein-Barr virus (EBV), establishes a chronic infection in its host by gaining access to the memory B-cell reservoir, where it persists undetected by the host's immune system. EBV encodes a membrane protein, LMP1, that appears to function as a constitutively active CD40 receptor, and is hypothesized to play a central role in EBV-driven differentiation of infected naive B cells to a memory B-cell phenotype. However, it has recently been shown that there is a critical role for CD40-CD40L interaction in B-cell immortalization by EBV (K.-I. Imadome, M. Shirakata, N. Shimizu, S. Nonoyama, and Y. Yamanashi, Proc. Natl. Acad. Sci. USA 100:7836-7840, 2003), indicating that LMP1 does not adequately recapitulate all of the necessary functions of CD40. The role of CD40 receptor expression on B cells for the establishment and maintenance of gammaHV68 latency is unclear. Data previously obtained with a competition model, demonstrated that in the face of CD40-sufficient B cells, gammaHV68 latency in CD40-deficient B cells waned over time in chimeric mice (I.-J. Kim, E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and M. A. Blackman, J. Immunol. 171:886-892, 2003). To further investigate the role of CD40 in gammaHV68 latency in vivo, we have characterized the infection of CD40 knockout (CD40(-/-)) mice. Here we report that, consistent with previous observations, gammaHV68 efficiently established a latent infection in B cells of CD40(-/-) mice. Notably, unlike the infection of normal C57BL/6 mice, significant ex vivo reactivation from splenocytes harvested from infected CD40(-/-) mice 42 days postinfection was observed. In addition, in contrast to gammaHV68 infection of C57BL/6 mice, the frequency of infected naive B cells remained fairly stable over a 3-month period postinfection. Furthermore, a slightly higher frequency of gammaHV68 infection was observed in immunoglobulin D (IgD)-negative B cells, which was stably maintained over a period of 3 months postinfection. The presence of virus in IgD-negative B cells indicates that gammaHV68 may either directly infect memory B cells present in CD40(-/-) mice or be capable of driving differentiation of naive CD40(-/-) B cells. A possible explanation for the apparent discrepancy between the failure of gammaHV68 latency to be maintained in CD40-deficient B cells in the presence of CD40-sufficient B cells and the stable maintenance of gammaHV68 B-cell latency in CD40(-/-) mice came from examining virus replication in the lungs of infected CD40(-/-) mice, where we observed significantly higher levels of virus replication at late times postinfection compared to those in infected C57BL/6 mice. Taken together, these findings are consistent with a model in which chronic virus infection of CD40(-/-) mice is maintained through virus reactivation in the lungs and reseeding of latency reservoirs.     

Characterization of Omental Immune Aggregates during Establishment of a Latent Gammaherpesvirus Infection

Herpesviruses are characterized by their ability to establish lifelong latent infection. The gammaherpesvirus subfamily is distinguished by lymphotropism, establishing and maintaining latent infection predominantly in B lymphocytes. Consequently, gammaherpesvirus pathogenesis is closely linked to normal B cell physiology. Murine gammaherpesvirus 68 (MHV68) pathogenesis in laboratory mice has been extensively studied as a model system to gain insights into the nature of gammaherpesvirus infection in B cells and their associated lymphoid compartments. In addition to B cells, MHV68 infection of macrophages contributes significantly to the frequency of viral genome-positive cells in the peritoneal cavity throughout latency. The omentum, a sheet of richly-vascularized adipose tissue, resides in the peritoneal cavity and contains clusters of immune cell aggregates termed milky spots. Although the value of the omentum in surgical wound-healing has long been appreciated, the unique properties of this tissue and its contribution to both innate and adaptive immunity have only recently been recognized. To determine whether the omentum plays a role in gammaherpesvirus pathogenesis we examined this site during early MHV68 infection and long-term latency. Following intraperitoneal infection, immune aggregates within the omentum expanded in size and number and contained virus-infected cells. Notably, a germinal-center B cell population appeared in the omentum of infected animals with earlier kinetics and greater magnitude than that observed in the spleen. Furthermore, the omentum harbored a stable frequency of viral genome-positive cells through early and into long-term latency, while removal of the omentum prior to infection resulted in a slight decrease in the establishment of splenic latency following intraperitoneal infection. These data provide the first evidence that the omentum is a site of chronic MHV68 infection that may contribute to the maintenance of chronic infection.     

An optimized CD8+ T-cell response controls productive and latent gammaherpesvirus infection

Strategies to prime CD8(+) T cells against Murine gammaherpesvirus 68 (gammaHV68; MHV68) latency have, to date, resulted in only limited effects. While early forms of latency (<21 days) were significantly reduced, effects were not seen at later times, indicating loss of control by the primed CD8(+) T cells. In the present study, we evaluated CD8(+) T cells in an optimized system, consisting of OTI T-cell-receptor (TCR) transgenic mice, which generate clonal CD8(+) T cells specific for K(b)-SIINFEKL of OVA, and a recombinant gammaHV68 that expresses OVA (gammaHV68.OVA). Our aim was to test whether this optimized system would result in more effective control not only of acute infection but also of later forms of latent infection than was seen with previous strategies. First, we show that OTI CD8(+) T cells effectively controlled acute replication of gammaHV68.OVA in liver, lung, and spleen at 8 and 16 days after infection of OTI/RAG mice, which lack expression of B and CD4(+) T cells. However, we found that, despite eliminating detectable acute replication, the OTI CD8(+) T cells did not prevent the establishment of latency in the OTI/RAG mice. We next evaluated the effectiveness of OTI T cells in OTI/B6 animals, which express B cells--a major site of latency in wild-type mice--and CD4(+) T cells. In OTI/B6 mice OTI CD8(+) T cells not only reduced the frequency of cells that reactivate from latency and the frequency of cells bearing the viral genome at 16 days after infection (similar to what has been reported before) but also were effective at reducing latency at 42 days after infection. Together, these data show that CD8(+) T cells are sufficient, in the absence of B cells and CD4(+) T cells, for effective control of acute replication. The data also demonstrate for the first time that a strong CD8(+) T-cell response can limit long-term latent infection.     

Immune control of the number and reactivation phenotype of cells latently infected with a gammaherpesvirus

Despite active immune responses, gammaherpesviruses establish latency. In a related process, these viruses also persistently replicate by using a mechanism that requires different viral genes than acute-phase replication. Many questions remain about the role of immunity in chronic gammaherpesvirus infection, including whether the immune system controls latency by regulating latent cell numbers and/or other properties and what specific immune mediators control latency and persistent replication. We show here that CD8(+) T cells regulate both latency and persistent replication and demonstrate for the first time that CD8(+) T cells regulate both the number of latently infected cells and the efficiency with which infected cells reactivate from latency. Furthermore, we show that gamma interferon (IFN-gamma) and perforin, which play no significant role during acute infection, are essential for immune control of latency and persistent replication. Surprisingly, the effects of perforin and IFN-gamma are site specific, with IFN-gamma being important in peritoneal cells while perforin is important in the spleen. Studies of the mechanisms of action of IFN-gamma and perforin revealed that perforin acts primarily by controlling the number of latently infected cells while IFN-gamma acts primarily by controlling reactivation efficiency. The immune system therefore controls chronic gammaherpesvirus infection by site-specific mechanisms that regulate both the number and reactivation phenotype of latently infected cells.     

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.     

Signaling through Toll-Like Receptors Induces Murine Gammaherpesvirus 68 Reactivation In Vivo

Murine gammaherpesvirus 68 (MHV68) establishes a lifelong infection in mice and is used as a model pathogen to study the role of viral and host factors in chronic infection. The maintenance of chronic MHV68 infection, at least in some latency reservoirs, appears to be dependent on the capacity of the virus to reactivate from latency in vivo. However, the signals that lead to MHV68 reactivation in vivo are not well characterized. Toll-like receptors (TLRs), by recognizing the specific patterns of microbial components, play an essential role in the activation of innate immunity. In the present study, we investigated the capacity of TLR ligands to induce MHV68 reactivation, both in vitro and in vivo. The stimulation of latently infected B cell lines with ligands for TLRs 3, 4, 5, and 9 enhanced MHV68 reactivation; the ex vivo stimulation of latently infected primary splenocytes, recovered from infected mice, with poly(I:C), lipopolysaccharide, flagellin, or CpG DNA led to early B-cell activation, B-cell proliferation, and a significant increase in the frequency of latently infected cells reactivating the virus. In vivo TLR stimulation also induced B-cell activation and MHV68 reactivation, resulting in heightened levels of virus replication in the lungs which correlated with an increase in MHV68-specific CD8⁺ T-cell responses. Importantly, TLR stimulation also led to an increase in MHV68 latency, as evidenced by an increase in viral genome-positive cells 2 weeks post-in vivo stimulation by specific TLR ligands. Thus, these data demonstrate that TLR stimulation can drive MHV68 reactivation from latency and suggests that periodic pathogen exposure may contribute to the homeostatic maintenance of chronic gammaherpesvirus infection through stimulating virus reactivation and reseeding latency reservoirs.     

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.