The human immunodeficiency virus (HIV) is the major etiological agent causing the Acquired Immunodeficiency Syndrome (AIDS). Transcription of HIV genes is a highly regulated process, and the regulatory mechanisms utilized by HIV seem to be novel. All primary HIV transcripts include a sequence, the transactivation response (TAR) element, which forms a stable stem-bulge-loop type of structure. The interaction of the TAR RNA element with the Tat protein is required for trans-activation of viral genes, thus this element has been described as an RNA enhancer. This RNA enhancer increases the levels of HIV transcripts by enhancing the processivity of the RNA pol II transcription complex that assembles on the HIV promoter. A 68,000dalton cellular protein (p68) binds to the TAR element the specificity for sequences in the loop. on the basis of the binding specificity it has been proposed that p68 regulates the Tat-TAR activity in vivo. The long-term objective of this proposal is to understand the mechanisms involved in HIV gene trans-activation. In particular we are interested in the roles played by the TAR element, the HIV Tat protein and the human p68 protein in regulating transcriptional trans-activation. The significance of this proposal is twofold: first, AIDS is a major public health threat in the USA and in the world, better understanding of the molecular biology of the virus is imperative. Second, the understanding of these novel mechanisms of transactivation of HIV genes will shed light on similar mechanisms of trans-activation of cellular genes.
The specific aims of this project are: First, to purify the p68 human protein to homogeneity. A specific and expedient assay to purify the protein has already been established. Second, to clone a cDNA encoding p68. Degenerate oligonucleotides will be used either in conventional screening methods or in PCR. Third, to establish the functional role of p68 in TAR dependent trans-activation.
