Understanding the latent replication mechanism of the Kaposi’ sarcoma associated herpesvirus.

Subhash Verma, Ph.D.

Kaposi’s sarcoma associated herpesvirus (KSHV) is the eighth human herpesvirus and is also called as HHV8. KSHV was initially discovered from the pigmented tumors (Kaposi’s sarcoma lesions) of AIDS patients but is now proven to be associated with the number of other lymphoproliferative diseases such as body cavity based lymphoma (a cancer of the lymphocyte) and multi-centric Castleman’s disease (severe enlargement of the lymph nodes). KSHV, like other members of the gammaherpesvirus family, infects humans and persists indefinitely in the infected cells by tethering to the host genome. KSHV reactivates and causes tumors in immune suppressed patients developed either by HIV infection or due to the administration of immunosuppressive drugs in organ transplant patients to prevent graft rejection. Incidence rate of KSHV tumors in HIV positive patients are very high and over 50% of the patients develop tumors. In organ transplant patient the incidence rate is relatively lower and approximately 9 per 100,000 person per year develop KSHV mediated tumors. In tumor cells virus persists as multicopy closed circular episomes with the expression of a repertoire of latent viral genes . These genes include Kaposin, viral Interferon Regulatory Factor (vIRF), viral Cyclin (vCyclin), vFLIP and Latency Associated Nuclear Antigen (LANA). Among these latent genes, LANA has been detected in every infected cell and is thus used as the signature molecule for the detection of KSHV infection. LANA is large nuclear protein and is detected in a punctate pattern on chromosomes of the infected cells. LANA is involved in episomal maintenance as well as transcriptional regulation of various cellular pathways to induce tumorigenesis. LANA is also involved in replication of the viral DNA during latent infection. Studies so far suggest that the Terminal Repeat (TR) of the viral genome serves as a replication origin site for the replication of the viral genome. KSHV genome posses multiple reiterated copies of the TR. Each TR unit is 801bp long high GC rich DNA element with two binding sites for LANA (LBS1/2). We have shown that LANA associates with the Origin Recognition complexes (ORC1-6) and the binding of LANA to the LBS1/2 recruits ORC1-6 to the TR and thus helps in replication initiation. Sequence upstream of the LBS1/2 is critical for replication initiation and is termed as replication element (RE). Recently, we have shown that RE and LBS1/2 containing plasmids replicate in LANA expressing cells once in a cell cycle manner. Expression of replication inhibitor geminin blocks replication of a plasmid containing KSHV replication origin. In order to identify therapeutic targets to block KSHV multiplication in the tumor cells, a better understanding of KSHV genome replication is warranted. Our current research interest is to understand the replication mechanism of the KSHV genome in the tumor cells. We are using a wide range of technique for the identification of replication initiation sites as well as the requirement and dynamics of cellular and viral proteins at the replication origins of the KSHV genome. AAmong these are the classic Meselson and Stahl density gradient experiment and a recently described Single Molecule Analysis of Replicated DNA (SMARD) technology. We are also using genetic approach for the identification and characterization of replication origins of the KSHV genome.

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