MECHANISM OF ACTION OF THE HIV REGULATORY PROTEINS

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Human immunodeficiency virus (HIV formerly HTLVIII/LAV/ARV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and associated diseases. In humans, HIV infection apparently displays a prolonged latent period with low levels of replication interspersed with bursts of viral replication and concomittant T cell death. Although the mechanism of viral latency is unknown, recent reports have implicated specific HIV gene products that are required for viral replication and thus may be essential for the shift from the latent to the cytopathic phase of viral infection. Our studies will focus on the biochemical and molecular genetic analysis of the two such trans-activator proteins, tat III (HTLV/III trans-activating gene) and art (anti- repression transactivator), and the modes of their regulation of HIV gene expression. For direct analysis of their target site binding and related studies, we have cloned the tat III and are in the process of cloning the art protein coding sequences in an efficient eukaryotic expression vector system using a baculovirus vector. The role of tat III binding to TAR will be studied using in vitro synthesized mRNAs in a cell free translation system. Similarly, the role of tat III in stabilization of mRNA will be studied using cell-free transcription system supplemented with HIV template DNA and cloned tat III protein. Similar reconstituted cell free translation and splicing systems, supplemented with art gene product, will analyze the mechanism of post-transcriptional regulation of the HIV gag and env gene transcripts by the art protein. Subsequent experiments will focus on site directed mutagenesis to determine the active sites of the cloned tat III and art proteins, and thus better define their mechanism of action. These studies therefore will delineate the biochemical and biophysical parameters of the two important viral regulatory proteins and thus help define their mechanisms of action in inducing the cytopathic phase of HIV infection in humans. The knowledge gained will be important in our designs to contain HIV infection and the progression of AIDS.