The HIV-1-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 efficiently mediates Tat-enhanced transcription (Tat function). Tat-SF contains P-TEFb, Spt5/Spt4, and Tat-SF1 and novel polypeptides, but none of the yeast coactivator SRB/MED-like proteins found associated with RNA Pol II holoenzyme. Notably, these Tat-SF cofactors can jointly complement the transcription initiation competent RNA Pol II holoenzyme for potent transcription elongation, reinitiation, and Tat transactivation, indicating that Tat-SF contains critical transcription factors. The mechanisms by which Tat-SF and RNA Pol II holoenzyme collectively mediate potent Tat-enhanced HIV-1 transcription are poorly understood. This proposal has three specific aims. The first will determine and compare the polypeptide composition of Tat-SF and Tat-SF cofactors derived from HeLa cytoplasm, nuclear extract, and nuclear pellet (chromatin).
The second aim will examine the mechanisms by which Tat-SF cofactors mediate potent Tat-enhanced HIV-1 transcription. These studies will provide insights into synergisms of Tat-SF cofactors within themselves and with other transcription factors and protein modifications (e.g. phosphorylation, acetylation, methylation) for potent Tat activation.
The third aim will specifically study the interplay of Tat-SF and the SRB/MED complex for efficient general transcription reinitiation and elongation. 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 efficient transcription. The effects of Tat on Tat-SF- and RNA Pol II holoenzyme-mediated transcription elongation and re-initiation will also be examined. Collectively, these studies will identify novel factors and mechanisms that are important for single and multiple round of HIV-1 transcription. They will also establish a unique well-defined in vitro system for future elaboration of the link between HIV-1 transcription elongation and mRNA processing either on naked or chromatin DNA templates. Together, understanding Tat-enhanced HIV-1 transcription at the molecular level will be instrumental for future therapeutic antiviral strategies to block viral replication.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM067538-01A2
Application #
6892475
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Tompkins, Laurie
Project Start
2005-02-01
Project End
2008-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
1
Fiscal Year
2005
Total Cost
$217,705
Indirect Cost
Name
Suny Downstate Medical Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
040796328
City
Brooklyn
State
NY
Country
United States
Zip Code
11203