Kaposi sarcoma-associated herpesvirus (KSHV/HHV8), the most recently discovered human herpesvirus, is the cause of Kaposi's sarcoma (KS), primary effusion lymphoma and some forms of Castleman's disease. KSHV-infected sequencing and expression studies, and have contributed to our understanding of genes critical to for cell transformation. Recent studies of oncoproteins form KSHV and other tumor viruses suggest that they may have evolved to inhibit innate immunity and promote virus survival. Some viral genes, however, are artificially dysregulated in tissue and little is known about which genes are active in KSHV-related tumors. Further, the effects of KSHV on cellular signaling pathways have not been fully explored. In the first part of this three-part proposal, we seek to examine KSHV gene transcription in tumors using specialized viral gene arrays. For some tumors, such as KS, single cells will be isolated by laser-assisted capture microdissection to generate cDNA libraries. This ensures sampling a homogenous transcriptosome rather than a mixture of expression states from different cells. In the second part, cellular transcriptional responses to KSHV vIL-6 and vIRF will be examined. Identifying pathways directly activated by vIL-6 will shed light on autocrine and paracrine responses to vIL-6 in lymphomas and Castleman's disease. Identifying cellular genes activated and repressed by vIRF will shed light on its cellular targets (the transcriptional co- adapters) acting in cellular transcription, particularly in response to interferon. Since vIRF causes cause cell transformation in vitro by activation of the cMYC oncogene, this will lead to important insights on how interferons control cell proliferation and inhibit tumor formation. In the final part, cell transcriptosome responses to linear and circular viral DNA, and to lytic virus replication will be examined We hypothesize that viral structures, such as the terminal repeats, may prevent p53 activation occurring from free DNA ends during lytic virus replication. These studies may identify cellular innate immunity sensor mechanisms activated during viral infection. To initiate studies immediately, commercially available membrane arrays will be used and program core slide array technologies will be used as they become available. Additional technical support for crafting arrays and bioinformatics will be provided from the program core. This also will allow us to develop collaborative interactions with the other program participants who are leading experts in their respective fields of herpes virology.
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