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.

A hallmark of productive infection by DNA viruses is the coupling of viral late gene expression to genome replication. Here we report the identification of open reading frame 30 (ORF30) and ORF34 as viral trans factors crucial for activating late gene transcription following viral DNA replication during lytic infection of murine gammaherpesvirus 68 (MHV-68). The mutant virus lacking either ORF30 or ORF34 underwent normal DNA replication but failed to express viral late gene transcripts, leading to nonproductive infection. In a reporter assay system, ORF30 and ORF34 were required for MHV-68 to activate the viral late gene promoters. Furthermore, studies using chromatin immunoprecipitation assays showed that the recruitment of RNA polymerase II to the viral late promoters during lytic infection was significantly reduced in the absence of ORF30 or ORF34. Together, the results suggest that ORF30 and ORF34 may play an important role in the assembly of the transcription initiation complex at the late gene promoters. Our discovery of the viral mutants that uncouple late gene transcription from DNA replication lays an important foundation to dissect the mechanism of this critical step of gene expression regulation.

Lytic replication of the tumor-associated human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus has important implications in pathogenesis and tumorigenesis. Herpesvirus lytic genes have been temporally classified as exhibiting immediate-early (IE), early, and late expression kinetics. Though the regulation of IE and early gene expression has been studied extensively, very little is known regarding the regulation of late gene expression. Late genes, which primarily encode virion structural proteins, require viral DNA replication for their expression. We have identified a murine gammaherpesvirus 68 (MHV-68) early lytic gene, ORF18, essential for viral replication. ORF18 is conserved in both beta-and gammaherpesviruses. By generating an MHV-68 ORF18-null virus, we characterized the stage of the virus lytic cascade that requires the function of ORF18. Gene expression profiling and quantitation of viral DNA synthesis of the ORF18-null virus revealed that the expression of early genes and viral DNA replication were not affected; however, the transcription of late genes was abolished. Hence, we have identified a gammaherpesvirus-encoded factor essential for the expression of late genes independently of viral DNA synthesis. Human gammaherpesviruses Epstein-Barr virus (EBV) andKaposi's sarcoma-associated herpesvirus (KSHV) contribute to the development of epithelial, hematopoietic, and endothelial cell cancers. EBV is associated with a number of malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and Hodgkin's disease (40). KSHV has been shown to be the causative agent of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma (9, 10, 51). A murine gammaherpesvirus, MHV-68, is a model to study gammaherpesvirus biology due to its conservation with EBV and KSHV in both genomic content and gene expression program. In addition, MHV-68 retains the ability to lytically infect various cell lines, including those of human origin, and provides a small-animal model for experimental gammaherpesvirus infection in vivo (36,46,52,55).Herpesviruses undergo both lytic and latent phases of infection. Following primary lytic infection in epithelial cells and lymphocytes, gammaherpesviruses establish a life-long latent infection, with intermittent bursts of lytic reactivation (33,35). This sporadic reactivation allows the virus to maintain a dynamic infectious reserve for transmission and secondary infection. During latent infection, the virus expresses a limited number of genes which promote the survival and proliferation of infected cells, resulting in transformation of a small percentage of cells (32). In KSHV, a subset of the transformed cell population supports spontaneous reactivation, leading to the expression of virally encoded cellular cytokine and chemokine homologues, including viral macrophage inflammatory proteins I, II,34,37). These viral cytokines have a paracrine growth-promoting effect on neighboring infected cells; thus, lyti...

The gamma subfamily of herpesviruses, including EpsteinBarr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), are associated with tumors and lymphoproliferative disorders (29,43). Murine gammaherpesvirus 68 (MHV-68) is phylogenetically related to EBV and KSHV and provides major research advantages by forming plaques on cell monolayers and establishing productive lytic and latent infections in mice (10,(64)(65)(66). Unlike EBV and KSHV, MHV-68 readily establishes productive infections in many cell culture systems and thus facilitates the examination of gammaherpesvirus replication and de novo infection (66,75,76). This provides an opportunity to examine cellular responses to de novo infection and their role in regulating gammaherpesvirus activity.Prostaglandins (PGs) are potent immunoregulatory lipid mediators generated by arachidonic acid metabolism via two cellular cyclooxygenases, constitutive COX-1 and inducible 22,26,61). PGs are formed by most cell types and exert a variety of actions in various tissues and cells via PG receptors (2,16,19,33,47). PGE 2 is an important proinflammatory prostanoid that mediates many symptoms of inflammation (41,53,56,70). Production of PGE 2 is catalyzed by COX-2, which is induced in response to factors such as bacterial lipopolysaccharides, mitogens, and cytokines (17,31,57,61). However, it is unknown what role the COX-2-PGE 2 pathway might play in responding to gammaherpesvirus de novo infection.Many viruses have been linked to the modulation of COX-2 expression and PG production (9,11,25,44,46,52,60,63,(77)(78)(79). COX-2 is responsible for the exaggerated biosynthesis of PGs under acute inflammatory conditions and in a diverse group of tumors (14,23,32,67). The COX-1 and COX-2 isozymes are the pharmacologic targets of nonsteroidal antiinflammatory drugs (NSAIDs) (26,50,61,71), and NSAIDs that block COX activity and PG production have been recognized as potentially effective antiviral therapeutics (4, 6-8, 51, 62, 73, 79). However, the effect of COX inhibitors on de novo infection of a gammaherpesvirus has not been previously examined. MHV-68 has been used as a model to study the efficacy of other antiviral compounds (3, 48, 49, 68), and we used this system to address the effects of COX-2 inhibition on gammaherpesvirus replication and infection.To enhance our understanding of virus-host cell interactions involved in the replication and pathogenesis of gammaherpesviruses, we examined MHV-68 de novo infection of NIH 3T3 and BHK-21 cells as a model for analyzing the role of PG production and COX-2 activity. We compared the effects of MHV-68 and UV-irradiated MHV-68 infection on COX-2 protein expression and COX-2 promoter activation. COX inhibitors {NS-398 [N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide] and indomethacin} were tested for their ability to suppress MHV-68 protein expression during de novo infection. Inhibition of protein expression and virion production by NS-398 was relieved in the presence of exogenous PGE 2 ,...

Kaposi's sarcoma-associated herpesvirus and murine gammaherpesvirus-68 (MHV-68) establish latent infections and are associated with various types of malignancies. They are members of the gamma-2 herpesvirus subfamily and encode a replication and transcriptional activator, RTA, which is necessary and sufficient to disrupt latency and initiate the viral lytic cycle in vitro. We have constructed a recombinant MHV-68 virus that overexpresses RTA. This virus has faster replication kinetics in vitro and in vivo, is deficient in establishing latency, exhibits a reduction in the development of a mononucleosis-like disease in mice, and can protect mice against challenge by wild-type MHV-68. The present study, by using MHV-68 as an in vivo model system, demonstrated that RTA plays a critical role in the control of viral latency and suggests that latency is a determinant of viral pathogenesis in vivo.