HIV-1 Tat protein is absolutely required for viral replication. Tat functions in concert with the cellular kinase cdk9 and human cyclinT1 (hCyclinT1) as a promoter-specific transcription elongation factor through binding to TAR in the 5'-untranslated leader of viral transcripts to stimulate processive transcription by RNA pol II. In this application, we propose to epitope map the entire surface of hCyclinT1 with human single-chain antibodies (sFv) to determine which epitopes on hCyclinT1 are suitable for the development of a new class of anti-HIV-1 therapeutics that target Tat-transactivation. Specifically, we will use our 15 billion-member human non-immune sFv phage-display library to isolate high-affinity human sFvs against all surface epitopes on hCyclinT1. These sFvs will be used to obtain a complete epitope map of hCyclinT1 through an analysis of their binding to hCyclinT1 polypeptides, truncation and point mutants and different species comparisons. BiaCore studies will be performed to obtain precise sFv affinity binding constants. We will also perform functional epitope mapping of hCyclinT1 by testing these same sFvs as anti-hCyclinT1 intrabodies to identify anti-hCyclinT1 intrabodies that selectively block Tat-mediated transactivation without disrupting the critical hCyclinT1-cdk9 interactions that are required for cellular gene transcription. We will determine if the anti-hCyclinT1 intrabodies that selectively inhibit Tat-mediated transactivation can also inhibit HIV-1 replication inhuman CD4+ peripheral blood T-cells transduced with a Tat-independent, self-inactivating (SIN) HIV-1vector to express the anti-hCyclinT1 intrabodies. We will also evaluate several normal cellular functions in stably transduced CD4+ T-cells that express anti-CyclinT1 intrabodies and determine if these functions are altered by the anti-hCyclinT1 intrabodies that exhibit anti-viral activity. Overall, the results of these studies will greatly aid in our immediate goal of targeting the Tat-transactivation mechanism in the design of small molecule drugs. Our long-term goal is to use anti-hCyclinT1 intrabodies together with anti-tat intrabodies in a clinical gene therapy trial.
