The gammaherpesvirus variably known as Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus eight (HHV8) is the likely etiologic agent of two malignancies, Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), that occur in patients with AIDS. In the majority of cells of these two KSHV-associated tumors the virus remains in a latent state, but in a subpopulation of cells KSHV is activated into lytic cycle gene expression. The applicants have found that a number of KSHV genes that are homologues of cellular chemokines, cytokines and anti-apoptotic factors that likely play critical roles in virus biology are expressed during the lytic phase of viral gene expression both in KS biopsies and in cell lines derived from PEL. As a seminal clue to the mechanism of lytic cycle activation we have identified a KSHV immediate-early gene product termed KSHV/Rta, partially encoded in ORF50, that activates expression of lytic cycle genes including vIL6 in PEL cells. Their global objective is to understand the role of the ORF50 gene product in the switch between latency and lytic cycle gene expression both in B cells and in microvascular endothelial cells. The investigators will analyze the events downstream of KSHV/Rta expression to determine whether ORF50/Rta is competent to activate the entire viral lytic cycle program. Using ribozyme inhibition the applicants will learn whether ORF50 is essential for reactivation of the virus. They will study the mechanism of action of KSHV/Rta, learning whether it acts directly, by binding DNA in the promoters of downstream genes, or indirectly recognizing DNA via other proteins. They will explore whether it autostimulates its own expression. They will delineate the pathway leading to activation of ORF50 expression, ultimately learning whether this activation is accompanied by acquisition or loss of cellular proteins that bind to the promoter of the ORF50 gene. They will determine whether the KSHV/ORF50 product plays an activating role in de novo infection of hematopoietic and endothelial cells or only functions during lytic cycle reactivation. The proposed experiments employ a combination of biologic, molecular, genetic and biochemical approaches to study a central feature of the KSHV life cycle. The studies should provide insights into molecular pathogenesis of KSHV-associated disease and address fundamental questions about control of oncogenic herpesvirus gene expression.
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