Kaposi's sarcoma-associated herpes virus (KSHVIHHV-8) is convincingly associated with Kaposi's sarcoma (KS), an endothelial cell tumor and B-cell lymphoproliferative diseases in immunosuppresssed individuals (AIDS patients and transplant recipients). Similar to other herpes viruses, KSHV lytic replication destroys the host cell, while the virus can persist indefinitely during latent infection. During latency in KS tumor cells and B cells fewer than 5% of KSHV genes are transcribed. We previously identified these genes in primary KS lesions and KSHV-associated lymphoma and found that every KSHV mRNA is unregulated during viral reactivation with the exception of the latency-associated-nuclear antigen (LANA). LANA is invariably expressed in every KS and PEL tumor cell. It is required for KSHV latent episome maintenance and it is able binds to the p53 and Rb tumor suppressor proteins, as well as GSK-31?, which implicates LANA in KSHV tumorigenesis. We have identified the promoter for LANA (LANAp), which regulates LANA, v-cyclin and v-FLIP, and this application proposes the detailed analysis of this promoter (Specific Aims 1 and 2). Since the LANAp is constitutively active in all KSHV latently infected cells, we hypothesize that LANAp activity depends on general and also B-lineage specific host cell factors. We propose to identify the binding sites for these factors in LANAp, identify the host factors and investigate their mechanism of action by a combination of biochemical approaches and BAC mutagenesis of the KSHV genome. We initiated our studies by showing that LANA itself auto-regulates LANAp, and hypothesize that this feedback loop is key to the establishment and maintenance of KSHV latency.
Specific aim 3 will test this hypothesis and determine in molecular detail the function of this regulatory loop. Understanding how LANA expression is regulated will have a significant impact on understanding KSHV latency and KS tumorigenesis. In the long term, our studies may yield targets for the development of small molecule that interfere with LANA, v-cyclin and v-FLIP expression, and thus have the potential to clear latent infection and to affect KS and PEL tumorigenesis.
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