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.     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

Turnover of T Cells in Murine Gammaherpesvirus 68-Infected Mice

Respiratory challenge of C57BL/6 mice with murine gammaherpesvirus 68 induces proliferation of T lymphocytes early after infection, as evidenced by incorporation of the DNA precursor bromodeoxyuridine. Using pulse-chase analysis, splenic and peripheral blood activated T lymphocytes were found to continue dividing for at least a month after the initial virus challenge. The results are in accord with the idea that T cells are stimulated for a substantial time after the acute, lytic phase of virus infection is resolved.     

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.     

Two Kinetic Patterns of Epitope-Specific CD8 T-Cell Responses following Murine Gammaherpesvirus 68 Infection

Murine gammaherpesvirus 68 (γHV68) provides an important experimental model for understanding mechanisms of immune control of the latent human gammaherpesviruses. Antiviral CD8 T cells play a key role throughout three separate phases of the infection: clearance of lytic virus, control of the latency amplification stage, and prevention of reactivation of latently infected cells. Previous analyses have shown that T-cell responses to two well-characterized epitopes derived from ORF6 and ORF61 progress with distinct kinetics. ORF6₄₈₇-specific cells predominate early in infection and then decline rapidly, whereas ORF61₅₂₄-specific cells continue to expand through early latency, due to sustained epitope expression. However, the paucity of identified epitopes to this virus has limited our understanding of the overall complexities of CD8 T-cell immune control throughout infection. Here we screened 1,383 predicted H-2^b-restricted peptides and identified 33 responses, of which 21 have not previously been reported. Kinetic analysis revealed a spectrum of T-cell responses based on the rapidity of their decline after the peak acute response that generally corresponded to the expression patterns of the two previously characterized epitopes. The slowly declining responses that were maintained during latency amplification proliferated more rapidly and underwent maturation of functional avidity over time. Furthermore, the kinetics of decline was accelerated following infection with a latency-null mutant virus. Overall, the data show that γHV68 infection elicits a highly heterogeneous CD8 T-cell response that segregates into two distinctive kinetic patterns controlled by differential epitope expression during the lytic and latency amplification stages of infection.Murine gammaherpesvirus 68 (γHV68) is a mouse pathogen closely related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV). Intranasal infection of mice with γHV68 leads to an acute infection in lung epithelial cells that is ultimately cleared and the concurrent establishment of latency in B cells, dendritic cells, and macrophages that undergoes amplification in the spleen and is maintained lifelong (11, 12). Even though γHV68 has the capacity to downregulate major histocompatibility complex class I (MHC-I) molecules (36), CD8 T cells specific for γHV68 are generated and have been shown to proliferate in response to cognate antigen, protect naive mice from γHV68 infection, lyse peptide-pulsed target cells in vivo and in vitro, and maintain the ability to produce antiviral cytokines (5, 6, 13, 27, 35). Until recently, knowledge of the antiviral CD8 T-cell repertoire in C57BL/6 mice was largely limited to two well-characterized epitopes derived from ORF6 and ORF61. T-cell responses to these epitopes have been shown to progress with distinct kinetics, with ORF6₄₈₇-specific cells predominating early in infection and ORF61₅₂₄-specific cells continuing to expand through early latency before declining and then persisting at higher levels late in infection (33). The difference in response kinetics correlates with the differential presentation of the epitopes, with the ORF6₄₈₇ epitope being expressed only during lytic infection and the ORF61₅₂₄ epitope being expressed both during lytic infection and during the latency amplification phase (22, 28). Additionally, the latency amplification phase is associated with the expansion of CD8 T cells with a Vβ4 T-cell receptor (TCR) component in several mouse strains (17), presumably due to a superantigen-like effect of the γHV68 M1 protein (4, 9).To better understand the breadth of the anti-γHV68 T-cell response, we used an enzyme-linked immunospot (ELISpot) approach to identify new epitopes. We identified a large number of epitopes derived from 26 proteins that drive the acute CD8 T-cell response to γHV68, which then narrowed over time, resulting in a limited antiviral response during latency. We did not observe inflation of any of the responses, as has been demonstrated for some murine cytomegalovirus (MCMV)-specific responses (20, 26). There was no evidence for functional exhaustion, as all detectable CD8 T-cell responses maintained functionality, but the responses declined in numbers over time. The decline in responses occurred over a broad kinetic range, which segregated into two general groups that correlated precisely with those previously described for ORF6 and ORF61. Thus, some responses declined rapidly after the acute phase of infection, while others declined more slowly.We examined two epitope-specific responses from each of the two patterns in detail over time for functional and phenotypic characteristics and found the responses to be highly heterogeneous, differing in TCR affinity, functional avidity, and proliferation rates. Importantly, slowly declining responses were not maintained as efficiently after infection with a latency-deficient virus, consistent with a role for epitope expression in driving the heterogeneous rate of decline in cell number after the acute infection. The data show that the response kinetics seen for the ORF6₄₈₇ and ORF61₅₂₄ responses are broadly applicable to multiple CD8 T-cell epitopes.