Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages

Intranasal infection of mice with the murine gamma-herpesvirus MHV-68 results in an acute lytic infection in the lung, followed by the establishment of lifelong latency. Development of an infectious mononucleosis-like syndrome correlates with the establishment of latency and is characterized by splenomegaly and the appearance of activated CD8+ T cells in the peripheral blood. Interestingly, a large population of activated CD8+ T cells in the peripheral blood expresses the V beta 4+ element in their TCR. In this report we show that MHV-68 latency in the spleen after intranasal infection is harbored in three APC types: B cells, macrophages, and dendritic cells. Surprisingly, since latency has not previously been described in dendritic cells, these cells harbored the highest frequency of latent virus. Among B cells, latency was preferentially associated with activated B cells expressing the phenotype of germinal center B cells, thus formally linking the previously reported association of latency gene expression and germinal centers to germinal center B cells. Germinal center formation, however, was not required for the establishment of latency. Significantly, although three cell types were latently infected, the ability to stimulate V beta 4+CD8+ T cell hybridomas was limited to latently infected, activated B cells.     

Dissecting the host response to a gamma-herpesvirus

The murine gamma-herpesvirus 68 (MHV-68) provides a unique experimental model for dissecting immunity to large DNA viruses that persist in B lymphocytes. The analysis is greatly facilitated by the availability of genetically disrupted (-/-) mice that lack key host-response elements, and by the fact that MHV-68 is a lytic virus that can readily be manipulated for mutational analysis. The mutant virus strategy is being used, for example, to characterize the part played in vivo by an MHV-68-encoded chemokine-binding protein that may ultimately find an application in human therapeutics. Experiments with various -/- mice and monoclonal antibody depletion protocols have shown very clearly that type I interferons (IFNs) are essential for the early control of MHV-68 replication, while CD4+ T cells producing IFN-gamma function to limit the consequences of viral persistence. Virus-specific CD8+ effectors acting in the absence of the CD4+ subset seem initially to control the lytic phase in the lung following respiratory challenge, but are then unable to prevent the reactivation of replicative infection in epithelia and the eventual death of CD4+ T-cell-deficient mice. This could reflect the fact that the interaction between the CD8+ T cells and the virus-infected targets is partially compromised by the MHV-68 K3 protein, which inhibits antigen presentation by MHC class I glycoproteins. Immunization strategies focusing on the CD8+ T-cell response to epitopes expressed during the lytic phase of MHV-68 infection can limit virus replication, but are unable to prevent the establishment of latency. Other experiments with mutant viruses also suggest that there is a disconnection between lytic MHV-68 infection and latency. The massive nonspecific immunoglobulin response and the dramatic expansion of Vbeta4+ CD8+ T cells, which is apparently MHC independent, could represent some sort of 'smoke screen' used by MHV-68 to subvert immunity. Although MHV-68 is neither Epstein-Barr virus nor human herpesvirus-8, the results generated from this system suggest possibilities that may usefully be addressed with these human pathogens. Perhaps the main lesson learned to date is that all the components of immunity are likely to be important for the control of these complex viruses.     

Signaling lymphocyte activation molecule-associated protein is a negative regulator of the CD8 T cell response in mice

The primary manifestation of X-linked lymphoproliferative syndrome, caused by a dysfunctional adapter protein, signaling lymphocyte activation molecule-associated protein (SAP), is an excessive T cell response upon EBV infection. Using the SAP-/- mouse as a model system for the human disease, we compared the response of CD8+ T cells from wild-type (wt) and mutant mice to various stimuli. First, we observed that CD8+ T cells from SAP-/- mice proliferate more vigorously than those from wt mice upon CD3/CD28 cross-linking in vitro. Second, we analyzed the consequence of SAP deficiency on CTL effector function and homeostasis. For this purpose, SAP-/- and wt mice were infected with the murine gamma-herpesvirus 68 (MHV-68). At 2 wk postinfection, the level of viral-specific CTL was much higher in mutant than in wt mice, measured both ex vivo and in vivo. In addition, we established that throughout 45 days of MHV-68 infection the frequency of virus-specific CD8+ T cells producing IFN-gamma was significantly higher in SAP-/- mice. Consequently, the level of latent infection by MHV-68 was considerably lower in SAP-/- mice, which indicates that SAP-/- CTL control this infection more efficiently than wt CTL. Finally, we found that the Vbeta4-specific CD8+ T cell expansion triggered by MHV-68 infection is also enhanced and prolonged in SAP-/- mice. Taken together, our data indicate that SAP functions as a negative regulator of CD8+ T cell activation.     

Pathogenesis of murine gammaherpesvirus infection in mice deficient in CD4 and CD8 T cells.

Murine gammaherpesvirus is a natural pathogen of wild mice. The virus infects alveolar cells and spleen cells during the primary infection and establishes a latent/persistent infection in B lymphocytes. Little is known about the immunological response to gammaherpesviruses during a primary infection. To address this issue, we investigated the pathogenesis of murine herpesvirus 68 (MHV-68) infection in mice deficient in CD4 or CD8 T-cell populations. Infection of the lung and spleen were greatly exacerbated in CD8-deficient mice, reflected by elevated virus titers in the lung and an increase in the number of infected splenocytes located around germinal centers. This finding contrasts with clearance of virus from the lung and spleen by day 12 postinfection in CD4-depleted animals. These data clearly indicate a major role for CD8 T cells in recovery from an acute MHV-68 infection. Whereas CD4 T cells fail to influence the course of infection in the lung, they do contribute to lymphoproliferation seen in the spleen (splenomegaly) during the primary infection. The significance of these results are discussed in relation to the immune response to other herpesviruses, in particular Epstein-Barr virus, with which MHV-68 shares similar molecular and biological properties.     

Differential gamma-herpesvirus distribution in distinct anatomical locations and cell subsets during persistent infection in mice

Murine gamma-herpesvirus 68 (MHV-68) provides an important experimental model for analyzing gamma-herpesvirus latent infection. After intranasal infection with MHV-68, we analyzed the distribution of the virus in different anatomical locations and purified populations of cells. Our data show that long-term latency is maintained in a variety of anatomical locations and cell populations with different frequencies. Importantly, we demonstrate that although latency in the lung is established in a variety of cell subsets, long-term latency in the lung is only maintained in B cells. In contrast, splenic latency is maintained in macrophages and dendritic cells, as well as in B cells. In blood, isotype-switched B cells constitute the major viral reservoir. These results show that the cell subsets in which latency is established vary within different anatomical sites. Finally, we demonstrate that long-term latency is accompanied by a low level of infectious virus in lung and spleen. These data have important implications for understanding the establishment and maintenance of latency by gamma(2)-herpesviruses.     

Apparent MHC-independent stimulation of CD8+ T cells in vivo during latent murine gammaherpesvirus infection.

Like EBV-infected humans with infectious mononucleosis, mice infected with the rodent gammaherpesvirus MHV-68 develop a profound increase in the number of CD8+ T cells in the circulation. In the mouse model, this lymphocytosis consists of highly activated CD8+ T cells strikingly biased toward V beta 4 TCR expression. Moreover, this expansion of V beta 4+CD8+ T cells does not depend on the MHC haplotype of the infected animal. Using a panel of lacZ-inducible T cell hybridomas, we have detected V beta 4-specific T cell stimulatory activity in the spleens of MHV-68-infected mice. We show that the appearance and quantity of this activity correlate with the establishment and magnitude of latent viral infection. Furthermore, on the basis of Ab blocking studies as well as experiments with MHC class II, beta2-microglobulin (beta2m) and TAP1 knockout mice, the V beta 4-specific T cell stimulatory activity does not appear to depend on conventional presentation by classical MHC class I or class II molecules. Taken together, the data indicate that during latent infection, MHV-68 may express a T cell ligand that differs fundamentally from both conventional peptide Ags and classical viral superantigens.     

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.     

Protective vaccination with hepatitis C virus NS3 but not core antigen in a novel mouse challenge model

BACKGROUND: Efficient vaccines against hepatitis C virus (HCV) infection are urgently needed. Vaccine development has been hampered by the lack of suitable small animal models to reliably test the protective capacity of immmunization. METHODS: We used recombinant murine gammaherpesvirus 68 (MHV-68) as a novel challenge virus in mice and tested the efficacy of heterologous candidate human vaccines based on modified vaccinia virus Ankara or adenovirus, both delivering HCV non-structural NS3 or core proteins. RESULTS: Recombinant MHV-68 expressing NS3 (MHV-68-NS3) or core (MHV-68-core) were constructed and characterized in vitro and in vivo. Mice immunized with NS3-specific vector vaccines and challenged with MHV-68-NS3 were infected but showed significantly reduced viral loads in the acute and latent phase of infection. NS3-specific CD8+ T cells were amplified in immunized mice after challenge with MHV-68-NS3. By contrast, we did neither detect a reduction of viral load nor an induction of core-specific CD8+ T cells after core-specific immunization. CONCLUSIONS: Our data suggest that the challenge system using recombinant MHV-68 is a highly suitable model to test the immunogenicity and protective capacity of HCV candidate vaccine antigens. Using this system, we demonstrated the usefulness of NS3-specific immunization. By contrast, our analysis rather discarded core as a vaccine antigen.     

Zinc Finger Antiviral Protein Inhibits Murine Gammaherpesvirus 68 M2 Expression and Regulates Viral Latency in Cultured Cells

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses by binding to specific viral mRNAs and repressing mRNA expression. Here we report that ZAP inhibits expression of murine gammaherpesvirus 68 (MHV-68) M2, which plays important roles in establishment and maintenance of viral latency. Downregulation of endogenous ZAP in cells harboring latent MHV-68 promoted lytic replication of the virus. These results suggest that ZAP inhibits M2 expression and regulates the maintenance of MHV-68 latency.     

IL-15-independent proliferative renewal of memory CD8+ T cells in latent gammaherpesvirus infection

IL-15 is known to be critical in the homeostasis of Ag-specific memory CD8(+) T cells following acute viral infection. However, little is known about the homeostatic requirements of memory CD8(+) T cells during a latent viral infection. We have used the murine gammaherpesvirus-68 (MHV-68) model system to investigate whether IL-15 is necessary for the maintenance of memory CD8(+) T cells during a latent viral infection. IL-15 is not essential either for the initial control of MHV-68 infection or for the maintenance of MHV-68-specific memory CD8(+) T cells. Even at 140 days postinfection, the proportion of CD8(+) T cells recognizing the MHV-68 epitopes were the same as in control mice. The maintenance of these memory CD8(+) T cells was attributable to their ability to turn over in vivo, probably in response to the presence of low levels of Ag. IL-15(-/-) mice had a significantly higher turnover rate within the virus-specific memory CD8(+) T cell population, which was the result of increased levels of viral gene expression rather than an increase in viral load. These cells did not accumulate in the spleens of the IL-15(-/-) mice due to an increased sensitivity to apoptosis as a result of decreased Bcl-2 levels. Intriguingly, memory CD8(+) T cells from latently infected mice failed to undergo homeostatic proliferation in a naive secondary host. These data highlight fundamental differences between memory CD8(+) T cells engaged in active immune surveillance of latent viral infections vs memory CD8(+) T cells found after acute viral infections.     

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.     

Murid herpesvirus 4 strain 68 M2 protein is a B-cell-associated antigen important for latency but not lymphocytosis

This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.     

Quantitative analysis of the acute and long-term CD4(+) T-cell response to a persistent gammaherpesvirus

The murine gammaherpesvirus 68 (MHV-68) replicates in respiratory epithelial cells, where it establishes a persistent, latent infection limited predominantly to B lymphocytes. The virus-specific CD4(+) T-cell response in C57BL/6 mice challenged intranasally with MHV-68 is detected first in the mediastinal lymph nodes and then in the cervical lymph nodes and the spleen. The numbers of MHV-68-specific CD4(+) T cells generated in congenic mice homozygous for disruption of the beta2-microglobulin gene tended to be higher, indicating that the absence of the CD8(+) set in this group resulted in a compensatory response. The peak frequency within the splenic CD4(+) T-cell population may reach 1:50 in the acute response; it then drops to 1:400 to 1:500 within 4 months and stays at that level in the very long term. Sorting for L-selectin (CD62L) expression established that all virus-specific CD4(+) T cells were initially CD62Llow, with >80% maintaining that phenotype for the next 14 months. The overall conclusion is that MHV-68-specific CD4(+) T cells remain activated (CD62Llow) and at a stable frequency in the face of persistent infection.     

Antibody-independent control of gamma-herpesvirus latency via B cell induction of anti-viral T cell responses

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine gamma-herpesvirus 68 (gammaHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell-/- mice during the early phase of gammaHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell-/- mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of gamma-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection.     

Identification of quantitative trait loci controlling activation of TRBV4 CD8+ T cells during murine gamma-herpesvirus-induced infectious mononucleosis

The murine gamma-herpesvirus, MHV-68, shares important biological and genetic features with the human gamma-herpesvirus, Epstein-Barr virus. Following intranasal infection, mice develop an infectious mononucleosis-like syndrome accompanied by increased numbers of activated CD8+ T cells in the blood. A consistent feature of the CD8+ T-cell activation is a marked increase in the frequency of cells expressing a TRBV4+ T-cell receptor. Previous studies suggested that the magnitude of TRBV4 expansion varied significantly among mouse strains, and was influenced by both MHC and non-MHC genes. Detailed analysis of strains with high (C57BL/6) or low (DBA/2) TRBV4 CD8+ T-cell expansion showed that differences in the degree of expansion were not a consequence of variation in genetic susceptibility to the viral infection. Rather, the magnitude of the TRBV4 CD8+ T-cell expansion correlated with differences in expression of the unidentified stimulatory ligand on activated, latently infected B cells. In the present study, analysis of TRBV4 expansion in C57BL/6, DBA/2, B6D2 F1 mice, BXD recombinant inbred strains, and the progeny of C57BL/6xDBA/2 F1 hybrids backcrossed to C57BL/6 demonstrated strong cumulative dominance of the low DBA/2 trait and moderately high heritability (h2 approximately 0.5). Two quantitative trait loci (QTLs) strongly associated with variance in TRBV4 expansion were identified using simple and composite mapping procedures. The first QTL is located on Chromosome (Chr) 17, near or proximal to H2. The second QTL is located on Chr 6 in a region spanning the Tcrb and Cd8a loci.     

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.     

Murine gammaherpesvirus targets type I IFN receptor but not type III IFN receptor early in infection

The innate immune response represents a primary line of defense against invading viral pathogens. Since epithelial cells are the primary site of gammaherpesvirus replication during infection in vivo and there are no information on activity of IFN-III signaling against gammaherpesviruses in this cell type, in present study, we evaluated the expression profile and virus-host interactions in mouse mammary epithelial cell (NMuMG) infected with three strains of murine gammaherpesvirus, MHV-68, MHV-72 and MHV-4556. Studying three strains of murine gammaherpesvirus, which differ in nucleotide sequence of some structural and non-structural genes, allowed us to compare the strain-dependent interactions with host organism. Our results clearly demonstrate that: (i) MHV-68, MHV-72 and MHV-4556 differentially interact with intracellular signaling and dysregulate IFN signal transduction; (ii) MHV-68, MHV-72 and MHV-4556 degrade type I IFN receptor in very early stages of infection (2–4 hpi), but not type III IFN receptor; (iii) type III IFN signaling might play a key role in antiviral defense of epithelial cells in early stages of murine gammaherpesvirus replication; (iv) NMuMG cells are an appropriate model for study of not only type I IFN signaling, but also type III IFN signaling pathway. These findings are important for better understanding of individual virus-host interactions in lytic as well as in persistent gammaherpesvirus replication and help us to elucidate IFN-III function in early events of virus infection.     

ZAP Inhibits Murine Gammaherpesvirus 68 ORF64 Expression and Is Antagonized by RTA

Zinc finger antiviral protein (ZAP) is an interferon-inducible host antiviral factor that specifically inhibits the replication of certain viruses, including HIV-1 and Ebola virus. ZAP functions as a dimer formed through intermolecular interactions of its N-terminal tails. ZAP binds directly to specific viral mRNAs and inhibits their expression by repressing translation and/or promoting degradation of the target mRNA. ZAP is not a universal antiviral factor, since some viruses grow normally in ZAP-expressing cells. It is not fully understood what determines whether a virus is susceptible to ZAP. We explored the interaction between ZAP and murine gammaherpesvirus 68 (MHV-68), whose life cycle has latent and lytic phases. We previously reported that ZAP inhibits the expression of M2, which is expressed mainly in the latent phase, and regulates MHV-68 latency in cultured cells. Here, we report that ZAP inhibits the expression of ORF64, a tegument protein that is expressed in the lytic phase and is essential for lytic replication. MHV-68 infection induced ZAP expression. However, ZAP did not inhibit lytic replication of MHV-68. We provide evidence showing that the antiviral activity of ZAP is antagonized by MHV-68 RTA, a critical viral transactivator expressed in the lytic phase. We further show that RTA inhibits the antiviral activity of ZAP by disrupting the N-terminal intermolecular interaction of ZAP. Our results provide an example of how a virus can escape ZAP-mediated immunity.