This proposal focuses on the protein--protein and protein--DNA interactions of a viral transcription factor, VP16, a herpes simplex virus virion component that, upon infection, is responsible for boosting expression of the viral immediate-early genes. The goals of this proposal are two-fold. First, they are directed at understanding in detail the protein--protein and protein--DNA interactions in which VP 16 participates, Interactions involving the human homeodomain protein Oct-1, another host cell protein termed HCF, and TAATGARAT cis-elements. Second, they are designed to determine whether analogous mechanisms operate during normal cellular transcription. The latter experiments will be guided by the results of recent studies which have shown that VPI6 can recognize the Oct-1 homeodomain, and that VP16 and Oct-1 bind adjacent cis-subelements within TAATGARAT sequences. The studies described in Specific Aim 1 employ footprinting and protein--DNA crosslinking techniques to identify the sequences recognized by Oct-1 and VP16 within TAATGARAT cis-elements. The studies described in Specific Aim 2 are aimed at understanding the structural basis of VP16 function. Among other techniques, they will employ site-directed mutagenesis to identify the regions of VP16 responsible for association with the Oct-1 homeodomain, HCF, and DNA. and test the hypothesis that an independent VP16 subdomain is responsible for Oct-1 homeodomain recognition.
Specific Aim 3 is concerned with the identity of HCF, presently known only as chromatographic fraction. HCF will be purified from mammalian and Drosophila extracts by a combination of protein-affinity and conventional chromatographic techniques. Alternatively, if purification proves difficult, cDNA's encoding HCF activity will be cloned by genetic complementation in yeast. Lastly, Specific Aim 4 is designed to test the hypothesis that interactions analogous to those of VP16 and the Oct- 1 homeodomain occur during normal cellular transcription. These studies will be based on the identification of the VP16 region involved in Oct-1 homeodomain association; if cellular transcription factors that function analogously to VPI6 exist, this homeodomain-recognition subdomain may have been conserved among them. Two strategies will be employed to exploit this potential conservation. First, PCR techniques will be utilized to identify similar protein-coding regions in cellular cDNA, and second, antisera raised against the VP16 homeodomain-recognition subdomain will be used to screen cDNA expression libraries. If these two strategies prove unsuccessful, an alternative approach using a novel """"""""fusion-phage"""""""" cDNA cloning technique will be employed to detect proteins that can interact with homeodomains.
