The HIV-l-encoded Tat protein is a potent transcriptional activator that is essential for viral replication. The long-term objective of this proposal is to gain a better understanding of the control of HIV-1 gene expression by Tat and cellular factors at the molecular level. This study will focus on a novel RNA Pol II complex (Tat- SF) that mediates Tat-enhanced transcription (Tat function). Tat-SF contains several factors (e.g., Spt5/Spt4, P-TEFb) and novel polypeptides, but none of the SRB/MED proteins. These Tat-SF cofactors are jointly required for general transcription and Tat function. This proposal has four specific aims. The first is to identify novel components of Tat-SF and examine their mechanisms of action. The polypeptide composition of the Tat-SF cofactors will be characterized by chromatographic methods and biochemical complementation assays. After the novel Tat-SF cofactors are identified by mass spectrometry and cloned, their interplay with other Tat-SF cofactors for Tat activity will be studied. The second will employ mutagenesis to determine the structure-function relationships of novel Tat-SF cofactors among themselves and with other transcription factors necessary for Tat function. The third will address the interplay of the Tat- SF cofactors and the SRB/MED complex for Tat activity. These experiments will include the detailed characterization of a potential SRB/MED complex-containing scaffold formed on the HIV-1 promoter that could be utilized by Tat-SF for transcription reinitiation. The effects of Tat and Tat-SF cofactors on Tat-SFand RNA Pol II holoenzyme-mediated reinitiation will be studied. Finally, the fourth aim will study the role of Tat in HIV-1 transcription on chromatin. A reconstituted system for Tat-dependent transcription on naked DNA will be optimized for Tat function on chromatin. The effects on chromatin of known factors necessary for transcription will be examined first, before further screening for potential activities that could complement the system for Tat function. Other studies will determine the effects of Tat, Tat-SF cofactors, and SRB/MED complex on transcription reinitiation on chromatin. These studies will establish a unique system for future elaboration of the link between HIV-1 transcription elongation and mRNA processing. Together, understanding Tat-enhanced HIV-1 transcription at the molecular level will be instrumental for future therapeutic antiviral strategies to block viral replication.
