The extent of HIV replication, including the switch from """"""""latency"""""""" in chronically infected cells, depends critically on the level of viral transcription that is effected by host and viral regulatory factors acting in conjunction with more general host transcription factors. The broad objective, critical for an understanding of virus replication and (ultimately) its control, is to detail the molecular mechanisms by which key viral and host factors productively activate the single promoter in the HIV LTR. The general approach is to reconstruct the regulatory events in biochemically-defined cell free systems, ultimately with completely homogeneous factors, and then to undertake more detailed studies of transcription mechanisms and structure-function relationships (using recombinant proteins) within the implicated factors. Such analyses will be complemented with related analyses of key factors (especially novel cellular cofactors) In various cellular assays, studies of possible variations in Tat target factors and cofactors In various cell types, and analysis of the mechanism of action of anti-Tat drugs. Related to these objectives the specific aims are: (1) to define the general transcription initiation and elongation factors involved in the assembly of functional initiation and elongation complexes on the minimal HIV promoter (TATA and Initiator elements), as well as the specific pathways involved and the structures of the corresponding intermediate complexes; (2) to investigate the role, including the mechanism of action, of known constitutive factors (Sp1, LBP-1) and novel negative cofactors in either elevating or restricting the basal transcription initiation or elongation functions of the general core promoter factors; (3) to identify biochemically purify, and obtain cDNAs that encode cellular cofactors essential for the function of the constitutive Sp1 and the viral-coded Tat activator on the HIV promoter in systems appropriately reconstituted with the other general factors; (4) to employ a variety of physical, functional, genetic and biochemical/immunological assays to determine the mechanism of action of key basal (Sp1) and regulatory (Tat) factors vis a vis interactions (alone or with specific cofactors) with specific targets in the general transcriptional machinery, including the basis for synergistic interactions between Tat and Sp1 and for the action of anti-Tat drugs; (5) to investigate structure-function relationships in general initiation and elongation factors (e.g., the multisubunit TATA-binding factor TFIID, the multisubunit promoter clearance factor TFIIH and its regulatory partner TFIIE), basal factors (e.g., LBP-1) and cofactors (e.g., Tat-binding) that are implicated as key targets for, or effectors of, Tat or Spl function. These studies are expected to provide a firm biochemical, structural and mechanistic basis for understanding key gene activation events in HIV replication in various cell types and for devising ways to block them.
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