Human herpesvirus 8 (HHV-8) is involved etiologically in HIV/AIDS-associated diseases Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease; in all, viral latent and lytic functions are believed to contribute. Molecular and mechanistic understanding of the virus-host interactions required for successful infection and replication is a necessary prerequisite for enabling the development of novel antiviral and therapeutic agents. HHV-8 encodes four viral interferon regulatory factors (vIRFs), all of which are expressed during lytic (productive) replication and three of which (vIRFs 1, 2 and 3) are known to be expressed, to varying degrees, in latently infected PEL cells and to support their viability. The vIRFs have been reported to interact in inhibitory fashion with various host-cell proteins, mainly involved in defense against virus infection. However, we and others have demonstrated empirically, via depletion and knockout studies, that vIRFs 2 and 3 suppress HHV-8 replication in PEL and other cell types. Although counterintuitive, such activity may contribute to overall viral fitness in natural infections, for example by limiting virus production to evade adaptive immune responses. On the other hand, vIRFs 1 and 4 have been found to promote lytic replication in culture; thus there is a balance between the replication-regulatory effects of the vIRFs. In the course of phenotypic studies of the vIRFs, we have identified vIRF-3 as a strong suppressor HHV-8 proteins RTA, required for latent-lytic switching and lytic replication, and viral interleukin-6 (vIL-6), involved in promoting lytic cycle and implicated in HHV-8- associated pathogenesis; these phenomena were identified in PEL and (model) iSLK epithelial cells, respectively. These findings may explain, at least in part, the observed replication-suppressive effects of vIRF- 3. Further to our published identification of vIRF-3:USP7 interaction as important for vIRF-3-mediated inhibition of HHV-8 replication and promotion of latently-infected PEL cell viability, we have determined that a USP7- binding motif-containing peptide derivative of vIRF-3 (v3pep) effects substantially enhanced vIRF-3 expression in latent/lytic PEL cells and lytically-infected iSLK cells and to suppression of lytic replication. The project proposed in this application will: (1) determine the mechanisms of vIRF-3 regulation of RTA and vIL-6, distinguishing between transcriptional and posttranscriptional effects and assessing hypothesized direct targeting by vIRF-3 of vIL-6/RTA promoter, or conceivably RNA, sequences; (2) examine the generality of vIRF- 3 regulation by v3pep in HHV-8-infected cells and involvement of vIRF-3 in the antiviral activity of the novel peptide. Data from this project will characterize mechanistically a newly identified function of vIRF-3 in lytic gene regulation and may provide the rationale and basis for future development of v3pep-based small-molecule antiviral age...