REGULATION OF CYTOKINES BY HIV1 TAT IN CENTRAL NERVOUS S

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The HIV-1 regulatory protein, Tat, has been shown to be essential for efficient viral replication and detrimental to brain cell populations. However, the mechanism whereby Tat induces neuropathology remains unknown. Tat is a transcriptional trans-activator of the HIV-1 LTR that interacts with the TAR RNA sequence located at the 5'sin end of the HIV-1 RNA. Recent studies have shown that LTR activation by Tat may require several cellular proteins including cyclin T1 and its partner cdk9, which associate with Tat and TAR and are positioned near the transcription start site. The interaction of these proteins allows for phosphorylation of RNA pol II, an event that increases the enzymatic activity of this protein. Tat also stimulates transcription of several immunomodulators and cytokines in CNS cells and it is thought that some of the CNS injury seen in AIDS brains is induced by soluble factors such as TNFalphaepsilon TGFbeta, IL1, and others. Since these genes lack the TAR RNA sequence, one could anticipate that the participation of an alternative pathway in Tat-mediated transcription of these genes. More recently, the PRINCIPAL INVESTIGATOR has identified a cellular protein, Puralpha, which exhibits the unique ability to bind to Tat and to interact with the GC/GA DNA sequence positioned upstream of a variety of cellular genes including TNFalpha and TGFbeta1. The PRINCIPAL INVESTIGATOR has proposed that the association of Puralpha with Tat alone or in combination with cyclin T1/cdk9 resulted in activation of TGFbeta1 and TNFalpha by Tat in CNS cells. The PRINCIPAL INVESTIGATOR proposes to examine the effect of Tat on the expression of TNFalpha and TGFbeta1 in primary CNS cultures and to examine the effect of cyclin T1/cdk9 and/or Puralpha in Tat-mediated transcriptional activation of these cytokines in primary cultures of human microglia and astrocytes. The PRINCIPAL INVESTIGATOR also proposes to examine the structural and functional interplay between Tat, cyclin T1/cdk9, and, Puralpha in CNS cells, and by constructing mutant proteins based on cyclin T1/cdk9 and Puralpha, develop therapeutic molecules that bind to Tat and sequester its activity. These studies will provide information concerning the mechanism of CNS cellular gene activation by Tat, and may serve as an initial step toward the development of targeted therapeutic compounds against HIV-1 Tat in the CNS.