Characterization of an Essential Gammaherpesvirus Lytic Gene

Vaithilingaraja Arumugaswami, UC Los Angeles

Individuals infected with HIV are at increased risk for developing specific types of malignancies. The human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are the etiological agents of some of the AIDS-related malignancies. EBV is associated with Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), and Hodgkin's disease (HD). KSHV causes multicentric Castleman's disease (MCD), primary effusion lymphoma (PEL) and an angiomatous skin cancer, Kaposi's sarcoma (KS) in HIV infected patients. Gammaherpesviruses undergo both lytic and latent forms of infection. Following primary lytic infection, to evade host immune response, the gammaherpesviruses establish latency in lymphocytes. An adventitious side effect of latent infection is transformation of infected cell. Lytic infection plays an important role in pathogenesis of gammaherpesvirus infection. Study of human gammaherpesvirus lytic infection is limited by the unavailability of cell line supporting robust infection. Murine gammaherpesvirus 68 (MHV-68) has been used as an animal model system to study gammaherpesvirus pathogenesis.

Through a signature-tagged mutagenesis study of the MHV-68 genome, we found a conserved lytic gene ORF18 which is essential for virus replication. Sequence comparison to the cellular protein database revealed no functional cellular homologue for ORF18. ORF18 is conserved among beta- and gamma-herpesviruses. There has been no detailed study on this essential gene ORF18. To understand the function of ORF18, I constructed an ORF18 null mutant (18S) using a MHV-68 BAC plasmid. The 18S is defective in replication, but can be rescued by exogenous expression of MHV-68 ORF18. Using the 18S virus, we characterized the stage of the virus lytic cascade requiring the function of ORF18. Viral 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 independent of viral DNA synthesis.

My hypothesis is that ORF18 is required for viral late gene expression by interacting with cellular and viral proteins. The overall goal of this proposal is to define the underlying mechanism of action of a tumor- associated gammaherpesvirus gene essential for expression of late lytic genes. In this proposal, I am interested in addressing the following important questions: 1) What microdomains of ORF18 are essential for its regulation of late lytic genes during virus replication? We have developed a high-throughput Mu-transposon mediated mutagenesis and genetic foot-printing technique which will be used to fine-map the essential microdomains of ORF18. 2) What are the cellular and viral proteins contributing to the function of ORF18 by protein-protein interaction? A yeast-two hybrid assay will be used to analyze ORF18 interacting protein partners. The results of this proposed study will provide further evidence regarding the function of this critical viral lytic protein ORF18 and the regulation of late lytic gene expression. Since ORF18 controls the expression of viral genes, the data obtained from microdomain analysis and interacting proteins will be used for developing small inhibitory molecules that will block ORF18 function, thus inhibiting the viral replication.