Gamma HV68 Research Articles

Age-dependent pathogenesis of murine gammaherpesvirus 68 infection of the central nervous system.

Gammaherpesvirus infection of the central nervous system (CNS) has been linked to various neurological diseases, including meningitis, encephalitis, and multiple sclerosis. However, little is known about the interactions between the virus and the CNS in vitro or in vivo. Murine gammaherpesvirus 68 (MHV-68 or (gamma)HV-68) is genetically related and biologically similar to human gammaherpesviruses, thereby providing a tractable animal model system in which to study both viral pathogenesis and replication. In the present study, we show the successful infection of cultured neuronal cells, microglia, and astrocytes with MHV-68 to various extents. Upon intracerebroventricular injection of a recombinant virus (MHV-68/LacZ) into 4-5-week-old and 9-10-week-old mice, the 4-5-week-old mice displayed high mortality within 5-7 days, while the majority of the 9-10-week-old mice survived until the end of the experimental period. Until a peak at 3-4 days post-infection, viral DNA replication and gene expression were similar in the brains of both mouse groups, but only the 9-10-week-old mice were able to subdue viral DNA replication and gene expression after 5 days post-infection. Pro-inflammatory cytokine mRNAs of tumor necrosis factor-alpha, interleukin 1beta, and interleukin 6 were highly induced in the brains of the 4-5-week-old mice, suggesting their possible contributions as neurotoxic factors in the agedependent control of MHV-68 replication of the CNS.

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

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

Murine gammaherpesvirus-68 elicits robust levels of interleukin-12 p40, but not interleukin-12 p70 production, by murine microglia and astrocytes.

Murine gammaherpesvirus-68 (gamma HV-68) is a tractable model to investigate the pathophysiology of human gammaherpesvirus infections, including Epstein-Barr virus (EBV). Herpesvirus infections are thought to play a role in the pathology of damaging, inflammatory diseases states of the central nervous system (CNS), such as multiple sclerosis. The ability of the host to mount a strong cell-mediated immune response is critical in determining the outcome of viral infections. Interleukin (IL)-12 is an important inflammatory cytokine that plays a pivotal role in the development of protective cell-mediated immune responses to viral infections. Given recent reports of associations between gammaherpesvirus infections and inflammatory disorders of the CNS, the authors investigated the ability of gamma HV-68 to induce the production of bioactive IL-12 in resident CNS cell types. In the present study, the authors demonstrate that gamma HV-68 infection is a potent stimulus for IL-12p40 production by murine microglia and astrocytes. However, despite the elevated expression of mRNA encoding IL-12p40 subunit, concomitant with robust secretion of IL-12p40 protein, gamma HV-68 failed to elicit the production of the bioactive IL-12p70 heterodimer. This failure did not result from an absence of T lymphocyte-derived signals or interactions between CNS cell types as determined by coculture studies. Taken together, these data suggest that the resident CNS cell types, astrocytes and microglia, are not significant sources of proinflammatory IL-12p70 in response to gammaherpesvirus infection. Indeed, the production of IL-12p40 may point to an anti-inflammatory role for these cells during herpesvirus infections of the CNS.

Function of Rta is essential for lytic replication of murine gammaherpesvirus 68.

Rta, encoded primarily by open reading frame 50, is well conserved among gammaherpesviruses. It has been shown that the Rta proteins of Epstein Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV-68; also referred to as gamma HV68) play an important role in viral reactivation from latency. However, the role of Rta during productive de novo infection has not been characterized in gammaherpesviruses. Since there are cell lines that can support efficient productive de novo infection by MHV-68 but not EBV or KSHV, we examined whether MHV-68 Rta plays a role in initiating viral lytic replication in productively infected cells. Rta, functioning as a transcriptional activator, can activate the viral promoter of early lytic genes. The amino acid sequence alignments of the Rta homologues suggest that the organizations of their functional domains are similar, with the DNA binding and dimerization domains at the N terminus and the trans-activation domain at the C terminus. We constructed two mutants of MHV-68 Rta, Rd1 and Rd2, with deletions of 112 and 243 amino acids from the C terminus, respectively. Rd1 and Rd2 could no longer trans-activate the promoter of MHV-68 gene 57, consistent with the deletions of their trans-activation domains at the C terminus. Furthermore, Rd1 and Rd2 were able to function as dominant-negative mutants, inhibiting trans-activation of wild-type Rta. To study whether Rd1 and Rd2 blocked viral lytic replication, purified virion DNA was cotransfected with Rd1 or Rd2 into fibroblasts. Expression of viral lytic proteins was greatly suppressed, and the yield of infectious viruses was reduced up to 10(4)-fold. Stable cell lines constitutively expressing Rd2 were established and infected with MHV-68. Transcription of the immediate-early gene, rta, and the early gene, tk, of the virus was reduced in these cell lines. The presence of Rd2 also led to attenuation of viral lytic protein expression and virion production. The ability of Rta dominant-negative mutants to inhibit productive infection suggests that the trans-activation function of Rta is essential for MHV-68 lytic replication. We propose that a single viral protein, Rta, governs the initiation of MHV-68 lytic replication during both reactivation and productive de novo infection.

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

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