Activation of Vav by the gammaherpesvirus M2 protein contributes to the establishment of viral latency in B lymphocytes

Gammaherpesviruses subvert eukaryotic signaling pathways to favor latent infections in their cellular reservoirs. To this end, they express proteins that regulate or replace functionally specific signaling proteins of eukaryotic cells. Here we describe a new type of such viral-host interaction that is established through M2, a protein encoded by murine gammaherpesvirus 68. M2 associates with Vav proteins, a family of phosphorylation-dependent Rho/Rac exchange factors that play critical roles in lymphocyte signaling. M2 expression leads to Vav1 hyperphosphorylation and to the subsequent stimulation of its exchange activity towards Rac1, a process mediated by the formation of a trimolecular complex with Src kinases. This heteromolecular complex is coordinated by proline-rich and Src family-dependent phosphorylated regions of M2. Infection of Vav-deficient mice with gammaherpesvirus 68 results in increased long-term levels of latency in germinal center B lymphocytes, corroborating the importance of the M2/Vav cross talk in the process of viral latency. These results reveal a novel strategy used by the murine gammaherpesvirus family to subvert the lymphocyte signaling machinery to its own benefit.     

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.     

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.     

Murine gammaherpesvirus 68 cyclin D homologue is required for efficient reactivation from latency

Murine gammaherpesvirus 68 (MHV68) is a gammaherpesvirus that was first isolated from murid rodents. MHV68 establishes a latent infection in the spleen and other lymphoid organs. Several gammaherpesviruses, including herpesvirus saimiri, human herpesvirus 8, and MHV68, encode proteins with extensive homology to the D-type cyclins. To study the function of the cyclin homologue, a recombinant MHV68 has been constructed that lacks the cyclin homologue and expresses beta-galactosidase as a marker (MHV68(cy-)). MHV68(cy-) grows in vitro with kinetics and to titers similar to those of the wild type. BALB/c mice infected with mixtures of equivalent amounts of the wild type and MHV68(cy-) show deficient growth of the MHV68(cy-) in an acute infection. Infection of SCID mice with virus mixtures also showed decreased MHV68(cy-) virus growth, indicating that the deficiency is not mediated by T or B cells. Although mice infected with mixtures containing 100 times as much MHV68(cy-) had greater splenic titers of the mutant virus than wild-type virus in acute infection, at 28 days postinfection splenocytes from these mice reactivated primarily wild-type virus. Quantitative PCR data indicate that equivalent genomes were present in the latent state. Reinsertion of the cyclin homologue into the cyclin-deleted virus restored the wild-type phenotype. These results indicate that the MHV68 cyclin D homologue mediates important functions in the acute infection and is required for efficient reactivation from latency.     

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.     

Global mRNA degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.     

De novo infection of B cells during murine gammaherpesvirus 68 latency

The mechanisms by which gammaherpesviruses maintain latency are unclear. Here we used a murine gammaherpesvirus model to show that previously uninfected B cells in immunocompetent mice can acquire virus during latency. In vivo depletion of T cells allowed viral reactivation, as measured by increased viral loads, but not enhanced transfer of virus to new cells. In the absence of both immune T cells and antibody following the transfer of latently infected cells into naïve animals, there was robust infection of new B cells. These data confirm that both T cells and antibody contribute to the control of gammaherpesvirus latency, reactivation, and spread.     

Immature and transitional B cells are latency reservoirs for a gammaherpesvirus

Gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68; also known as gammaherpesvirus 68 [γHV68] or murine herpesvirus 4 [MuHV-4]), establish lifelong latency in the resting memory B cell compartment. However, little is known about how this reservoir of infected mature B cells is maintained for the life of the host. In the context of a normal immune system, the mature B cell pool is naturally maintained by the renewable populations of developing B cells that arise from hematopoiesis. Thus, recurrent infection of these developing B cell populations could allow the virus continual access to the B cell lineage and, subsequent to differentiation, the memory B cell compartment. To begin to address this hypothesis, we examined whether MHV68 establishes latency in developing B cells during a normal course of infection. In work described here, we demonstrate the presence of viral genome in bone marrow pro-pre-B cells and immature B cells during early latency and immature B cells during long-term latency. Further, we show that transitional B cells in the spleen are latently infected and express the latency-associated nuclear antigen (LANA) throughout chronic infection. Because developing B cells normally exhibit a short life span and a high rate of turnover, these findings suggest a model in which gammaherpesviruses may gain access to the mature B cell compartment by recurrent seeding of developing B cells.     

Maintenance of motor neuron progenitors in Xenopus requires a novel localized cyclin

The ventral spinal cord contains a pool of motor neuron progenitors (pMNs), which sequentially generate motor neurons and oligodendrocytes in the embryo. The mechanisms responsible for the maintenance of pMNs are not clearly understood. We have identified a novel cyclin, cyclin Dx (ccndx), which is specifically expressed in pMNs in Xenopus. Here, we show that inhibition of ccndx causes paralysis in embryos. Furthermore, we show that maintenance of pMNs requires ccndx function. In addition, inhibition of ccndx results in the specific loss of differentiated motor neurons. However, the expression of interneuron or sensory neuron markers is unaffected in these embryos, suggesting that the role of ccndx is specifically to maintain pMNs. Thus, we have identified, for the first time, a tissue-specific cell-cycle regulator that is essential for the maintenance of a pool of neural progenitors in the vertebrate spinal cord.     

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 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.     

Maintenance of peritoneal B-1a lymphocytes in the absence of the spleen

Positive selection by autoantigens is believed to play an important role in the generation/maintenance of B-1a cells. Recently, it has been described that splenectomy results in the loss of an already established B-1a cell pool. To elucidate whether the spleen influences the peritoneal B-1a repertoire, we have analyzed the consequences of splenectomy in the recently established IgL-transgenic L2 mouse model. L2 mice are characterized by a severe block of B-2 development and predominance of B-1a cells, which exhibit a pronounced IgH oligoclonality, presumably due to positive selection by autoantigens. In this study, we show that, in striking contrast to splenectomized normal mice, L2 mice exhibit unchanged frequencies of peritoneal B-1a cells. The IgH repertoire of these B-1a cells, however, was severely perturbed in that the previously described predominant B-1a H chains were no longer present. The repertoire changes were partial since phosphatidylcholine-specific B-1a cells were present in similar numbers before and after splenectomy. Thus, splenic Ags appear to act as "survival factors" for major subsets of peritoneal B cells. The loss of B-1a cells in the absence of such factors is compensated by repertoire changes among B-1a cells in B cell lymphopenic L2 but not normal mice.     

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.     

Perturbation of B cell activation in SLAM-associated protein-deficient mice is associated with changes in gammaherpesvirus latency reservoirs

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)) interactions with SLAM family proteins play important roles in immune function. SAP-deficient mice have defective B cell function, including impairment of germinal center formation, production of class-switched Ig, and development of memory B cells. B cells are the major reservoir of latency for both EBV and the homologous murine gammaherpesvirus, gammaherpesvirus 68. There is a strong association between the B cell life cycle and viral latency in that the virus preferentially establishes latency in activated germinal center B cells, which provides access to memory B cells, a major reservoir of long-term latency. In the current studies, we have analyzed the establishment and maintenance of gammaHV68 latency in wild-type and SAP-deficient mice. The results show that, despite SAP-associated defects in germinal center and memory B cell formation, latency was established and maintained in memory B cells at comparable frequencies to wild-type mice, although the paucity of memory B cells translated into a 10-fold reduction in latent load. Furthermore, there were defects in normal latency reservoirs within the germinal center cells and IgD(+)"naive" B cells in SAP-deficient mice, showing a profound effect of the SAP mutation on latency reservoirs.     

Role for MyD88 signaling in murine gammaherpesvirus 68 latency

Toll-like receptors (TLRs) are known predominantly for their role in activating the innate immune response. Recently, TLR signaling via MyD88 has been reported to play an important function in development of a B-cell response. Since B cells are a major latency reservoir for murine gammaherpesvirus 68 (MHV68), we investigated the role of TLR signaling in the establishment and maintenance of MHV68 latency in vivo. Mice deficient in MyD88 (MyD88(-/-)) or TLR3 (TLR3(-/-)) were infected with MHV68. Analysis of splenocytes recovered at day 16 postinfection from MyD88(-/-) mice compared to those from wild-type control mice revealed a lower frequency of (i) activated B cells, (ii) germinal-center B cells, and (iii) class-switched B cells. Accompanying this substantial defect in the B-cell response was an approximately 10-fold decrease in the establishment of splenic latency. In contrast, no defect in viral latency was observed in TLR3(-/-) mice. Analysis of MHV68-specific antibody responses also demonstrated a substantial decrease in the kinetics of the response in MyD88(-/-) mice. Analysis of wild-type x MyD88(-/-) mixed-bone-marrow chimeric mice demonstrated that there is a selective failure of MyD88(-/-) B cells to participate in germinal-center reactions as well as to become activated and undergo class switching. In addition, while MHV68 established latency efficiently in the MyD88-sufficient B cells, there was again a ca. 10-fold reduction in the frequency of MyD88(-/-) B cells harboring latent MHV68. This phenotype indicates that MyD88 is important for the establishment of MHV68 latency and is directly related to the role of MyD88 in the generation of a B-cell response. Furthermore, the generation of a B-cell response to MHV68 was intrinsic to B cells and was independent of the interleukin-1 receptor, a cytokine receptor that also signals through MyD88. These data provide evidence for a unique role for MyD88 in the establishment of MHV68 latency.