Comprehensive analysis of KSHV gene expression
Yoshihiro Izumiya, University of California, Davis
Kaposi's sarcoma (KS) is the major malignancy occurring in AIDS patients. The etiologic factor for KS has been linked to a human herpesvirus, called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV DNA is present in virtually all tumors. KSHV exists in two phases in its host; these are the latent state and the lytic replication state. In the latent state only restricted viral genes are transcribed, while in the lytic state more than eighty viral genes are transcribed in cascade fashion. Many AIDS patients are life-long carriers of latent virus, the reactivations of which are unpredictable. Most of the spindle cells in KS tumors are latently infected with the virus, implicating a direct involvement of viral latent genes in the transformation of KS cells. However, KSHV lytic replication also contributes significantly to the formation of KS lesions, by either facilitating viral spread to the target site or releasing paracrine factors to support the growth of the neighboring KS tumor cells. Similarly, the KSHV genome is frequently detected in HIV-associated primary effusion lymphoma (PEL) biopsies, and all cell lines derived from PEL harbor KSHV, with varying degrees of expression of viral latent and lytic genes. It thus appears that both classes of genes contribute to the oncogenic processes.
A single KSHV immediate-early gene product, K-Rta, is sufficient to induce lytic reactivation of the latent KSHV genome in BCBL-1 cells, a cell line model for PEL. A large number of K-Rta responsive KSHV promoters have been identified. K-Rta also induces several cellular gene expressions such as IL-6, CD21 and CD23, which are believed to contribute to pathogenesis of this virus. However, much needs to be learned about the mechanism(s) of these processes. Understanding the molecular mechanisms of regulation of KSHV gene expression and disruption of latency should greatly aid in the therapy and prevention of disease associated with this virus. This research proposal, entitled "Comprehensive analysis of KSHV gene expression." is designed to investigate a "dynamic" picture of KSHV gene expression based on genome-wide analysis of KSHV gene regulation with a focus on K-Rta.
Through the experimental activities in the following two specific aims, we will build a model of the KSHV reactivation process. Results from these experiments will enable the development of a comprehensive program to analyze the assembly of transcriptional factors at viral promoters, and hence, to define the detailed mechanism of viral gene transcription and reactivation.
Aim1: Transactivation mechanism(s) of K-Rta.
(a) Identification of authentic K-Rta target promoters and the temporal dynamics of K-Rta binding to these sites.
(b) Genome-wide map of nucleosome occupancy, acetylation and methylation of the KSHV chromatin during reactivation.
Aim2: Effects of cellular cofactor on K-Rta: Mechanism of NF-kB mediated K-Rta repression.
(a) Interplay between NF-kB and RBP-Jk on KSHV genome.
(b) Virological significance of K-Rta cofactor