COX-2 induction during murine gammaherpesvirus 68 infection leads to enhancement of viral gene expression

The murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) model provides many advantages for studying virus-host interactions involved in gammaherpesvirus replication, including the role of cellular responses to infection. We examined the effects of cellular cyclooxygenase-2 (COX-2) and its by-product prostaglandin E(2) (PGE(2)) on MHV-68 gene expression and protein production following de novo infection of cultured cells. Western blot analyses revealed an induction of COX-2 protein in MHV-68-infected cells but not in cells infected with UV-irradiated MHV-68. Luciferase reporter assays demonstrated activation of the COX-2 promoter during MHV-68 replication. Two nonsteroidal anti-inflammatory drugs, a COX-2-specific inhibitor (NS-398) and a COX-1-COX-2 inhibitor (indomethacin), substantially reduced MHV-68 protein production in infected cells. Inhibition of viral protein expression and virion production by NS-398 was reversed in the presence of exogenous PGE(2). Global gene expression analysis using an MHV-68 DNA array showed that PGE(2) increased production of multiple viral gene products, and NS-398 inhibited production of many of the same genes. These studies suggest that COX-2 activity and PGE(2) production may play significant roles during MHV-68 de novo infection.     

Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome

Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) in Escherichia coli. During propagation in E. coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked by loxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAC-cloned MHV-68. Growth of the BAC-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.     

CD28(-/-) mice show defects in cellular and humoral immunity but are able to control infection with murine gammaherpesvirus 68

The role of CD28-dependent costimulatory interactions in the development and maintenance of antiviral immune responses was investigated in a mouse model of gammaherpesvirus infection. CD28(-/-) mice could clear a productive infection with murine gammaherpesvirus 68 (MHV-68), although early lung viral titers were significantly increased. Both CD28(-/-) and CD28(+/+) mice maintained effective long-term control of MHV-68. Gamma interferon responses appeared to develop more slowly in CD28(-/-) mice, while cytotoxic T-cell activity was similar to that in wild-type mice. Splenomegaly developed normally in CD28(-/-) mice, whereas virus-specific antibody responses were significantly reduced and aberrant class switching was observed. This work demonstrates that costimulatory interactions involving CD28 are not an absolute requirement for the control of infection with MHV-68.     

Murine gammaherpesvirus 68 lacking thymidine kinase shows severe attenuation of lytic cycle replication in vivo but still establishes latency

The lytic cycle functions of gammaherpesviruses have received relatively little attention to date, at least in part due to the lack of a convenient experimental model. The murine gammaherpesvirus 68 (MHV-68) now provides such a model and allows the roles of individual lytic cycle gammaherpesvirus proteins to be evaluated in vivo. We have used MHV-68 to determine the contribution of a gammaherpesvirus thymidine kinase (TK) to viral lytic replication and latency establishment. MHV-68 mutants with a disrupted TK gene grew normally in vitro but showed a severe attenuation of replication in the lungs after intranasal inoculation, with lytic titers at least 1,000-fold lower than those of wild-type and revertant viruses. Nevertheless, the establishment of latency by the TK-deficient mutants, while delayed, was not prevented by their lytic replication deficit. The viral TK clearly plays a crucial role in the capacity of MHV-68 to replicate efficiently in its natural host but does not seem to be essential to establish a persistent infection. The potential of TK-deficient mutants as gammaherpesvirus vaccines is discussed.     

Identification of cis sequences required for lytic DNA replication and packaging of murine gammaherpesvirus 68

Human gammaherpesviruses are associated with lymphomas and other malignancies. Murine gammaherpesvirus 68 (MHV-68) infection of mice has emerged as a model for understanding gammaherpesvirus pathogenesis in vivo. In contrast to human gammaherpesviruses, MHV-68 replicates in permissive cell lines in a robust manner, presenting an efficient model to study the basic mechanisms for DNA replication and recombination processes. In addition, MHV-68 also infects a broad range of cells of different tissue types and from different host species, and the viral genome persists as an episome in infected cells. These features make MHV-68 an attractive system on which to build gene delivery vectors. We have therefore undertaken a study to identify the cis elements required for MHV-68 genome replication and packaging. Here we report that an 8.4-kb MHV-68 genomic fragment between ORF66 and ORF73 conferred on the plasmid the ability to replicate; replication required the presence of either de novo viral infection or viral reactivation from latency. We further mapped the origin of lytic replication (oriLyt) to a 1.25-kb region. Moreover, we demonstrated that the terminal repeat of the viral genome is sufficient for packaging of the replicated oriLyt plasmid into mature viral particles. Functional identification of the MHV-68 oriLyt and packaging signal has laid a foundation for investigating the mechanisms controlling gammaherpesvirus DNA replication during the viral lytic phase and will also serve as a base on which to design gene delivery vectors.     

Persistent gammaherpesvirus replication and dynamic interaction with the host in vivo

Gammaherpesviruses establish life-long persistency inside the host and cause various diseases during their persistent infection. However, the systemic interaction between the virus and host in vivo has not been studied in individual hosts continuously, although such information can be crucial to control the persistent infection of the gammaherpesviruses. For the noninvasive and continuous monitoring of the interaction between gammaherpesvirus and the host, a recombinant murine gammaherpesvirus 68 (MHV-68, a gammaherpesvirus 68) was constructed to express a firefly luciferase gene driven by the viral M3 promoter (M3FL). Real-time monitoring of M3FL infection revealed novel sites of viral replication, such as salivary glands, as well as acute replication in the nose and the lung and progression to the spleen. Continuous monitoring of M3FL infection in individual mice demonstrated the various kinetics of transition to different organs and local clearance, rather than systemically synchronized clearance. Moreover, in vivo spontaneous reactivation of M3FL from latency was detected after the initial clearance of acute infection and can be induced upon treatment with either a proteasome inhibitor Velcade or an immunosuppressant cyclosporine A. Taken together, our results demonstrate that the in vivo replication and reactivation of gammaherpesvirus are dynamically controlled by the locally defined interaction between the virus and the host immune system and that bioluminescence imaging can be successfully used for the real-time monitoring of this dynamic interaction of MHV-68 with its host in vivo.     

Coordinate regulation of DNA damage and type I interferon responses imposes an antiviral state that attenuates mouse gammaherpesvirus type 68 replication in primary macrophages

DNA damage response (DDR) is a sophisticated cellular network that detects and repairs DNA breaks. Viruses are known to activate the DDR and usurp certain DDR components to facilitate replication. Intriguingly, viruses also inhibit several DDR proteins, suggesting that this cellular network has both proviral and antiviral features, with the nature of the latter still poorly understood. In this study we show that irradiation of primary murine macrophages was associated with enhanced expression of several antiviral interferon (IFN)-stimulated genes (ISGs). ISG induction in irradiated macrophages was dependent on type I IFN signaling, a functional DNA damage sensor complex, and ataxia-telangiectasia mutated kinase. Furthermore, IFN regulatory factor 1 was also required for the optimal expression of antiviral ISGs in irradiated macrophages. Importantly, DDR-mediated activation of type I IFN signaling contributed to increased resistance to mouse gammaherpesvirus 68 replication, suggesting that the coordinate regulation of DDR and type I IFN signaling may have evolved as a component of the innate immune response to virus infections.     

Murine gammaherpesvirus 68 open reading frame 11 encodes a nonessential virion component

Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.     

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.     

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.     

Murine gammaherpesvirus 68 contains two functional lytic origins of replication

A 1.25-kbp DNA fragment from the right side of the genome containing the lytic origin of replication (oriLyt) of murine gammaherpesvirus 68 (MHV-68) has been identified by a plasmid replication assay. Here we show that a mutant MHV-68 with a deletion of an essential part of this oriLyt, generated by using an MHV-68 bacterial artificial chromosome, was only slightly attenuated and still able to replicate but that a mutant containing an additional deletion on the left side of the genome was replication deficient. The newly identified region was sufficient to support plasmid replication, thus providing evidence for a second oriLyt.