) Kaposi sarcoma associated herpesvirus (KSVH or HHV-8) is the probable causative etiological agent of all forms of KS. Unlike most herpesviruses, infection with KSHV is very rare in most human populations except in Central Africa and the Mediterranean, but it is very common in male homosexual AIDS patients, who also have high rates of KS lesions. The genomes are found in most or all KS spindle and endothelial cells, as well as in Primary Effusion Lymphoma and Multicentric Castleman's Disease tumor cells in AIDS patients. Only the viral nuclear proteins LANA and vCYCD are known to be expressed in all HHV-8 associated tumor cells in vivo, but the hypervariable ORF-K1 and ORF-K15 membrane proteins that we have found to be encoded at the extreme LHS and RHS ends of the genome are also candidate oncogenes or latency regulators. We have identified four distinct subtypes of the Ig receptor-like ORF-K1 protein and two very different forms (or alleles ) of the complex spliced ORF-K15 gene. ORF-K1 is a highly glycosylated membrane signaling protein, that produces transformed foci in DNA-transfected cells and substitutes for the STP oncogene in HVS tumorigene- sis assays. ORF-K15 is a highly diverged orthologue of the EBV LMP2 gene and appears to encode a latent state integral membrane signaling protein. In this project we plan to (1) Determine the patterns of expression of ORF-K15 mRNA and protein in KS lesions and PEL tumor cells; (2) Ask whether there are distinctive or alternative latent and lytic promoter pathways for ORF-K15 P and M gene regulation; (3) Evaluate the functional activity of the two conserved YXXL motifs in the ORF-K15 cytoplasmic tail in lymphoid cell signaling assays and phosphotyrosine kinase interactions; (4) Determine whether Tet-inducible intact ORF-K15 alters cell growth or proliferation in focus forming assays and whether it blocks B cell activation or signaling and EBV or HHV8 reactivation, and (5) Initiate long-term genetic studies to generate ORF-K15 deleted HHV8 for directly assessing its role in latency and transformation.
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