Understanding the mechanism of KSHV latent DNA replication

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DESCRIPTION (provided by applicant): Long-term goals: The overall goal of this project is to elucidate the replication mechanism of KSHV during latent infection. Kaposi's sarcoma associated Herpesvirus (KSHV) predominantly infects B cells and endothelial cells and persists indefinitely with the expression of a limited number of genes. These latent genes have been shown to induce cell proliferation/tumorigenesis. Viral DNA which persists as an episome gets passage to the dividing tumor cells during tumorigenesis. Although a number of studies have shown that TR region of the genome can support replication, the mechanism of the replication is not clearly understood. Studies elucidating the mechanism of latent DNA replication will provide clues of viral and cellular molecules required for replication initiation and therefore will allow targeting of these molecules as potential therapeutics. KSHV associated tumors is a major health problem for the HIV infected individuals as well as Organ transplanted patients undergoing immunosuppressive therapies to prevent graft rejections. Specific aims:1- Identification of the replication initiation sites on the KSHV genome and cloning of the KSHV regions capable of replicating and their replicative potential in Dpnl sensitivity assay. 2- Evaluation of the role of trans acting viral protein LANA on the replication of KSHV genome fragment. Meselson and Stahl experiment with the plasmids containing these replicating elements to determine the rate and nature (semi- conservative) of replication by these replicons. Evaluation for long-term persistence by these plasmids. 3- Determination of the cellular protein dynamics at the replication origins. The candidate has a Ph.D. in Biotechnology and almost four years of post-doctoral research experience in molecular virology working on KSHV encoded LANA and the replication of KSHV genome during latency. The immediate goal of the candidate is to understand the mechanism of KSHV latent DNA replication more specifically mapping of the LANA independent replication origin, sequence requirement and protein dynamics at replication initiation sites to specifically block KSHV mediated cancers. My long-term career goal is to establish myself as a independent researcher in a an academic environment in the field of molecular virology. This research will help to understand the replication and passage of virus in tumor cells and thereby using specific inhibitors replication and passage of the virus in the tumor cells will be blocked.