The emergence of drug-resistant HIV-1 strains presents a challenge for the design of new drugs. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to overcome the resistance of HIV-1 to anti-viral agents. The proposed research is designed to delineate the role of host cell-cycle dependent kinase 2 (CDK2) in the regulation of HIV-1 transcription mediated by HIV-1 Tat protein. HIV-1 Tat stimulates elongation of viral transcription by binding to CDKg/cyclin T1 and inducing hyperphosphorylation of the C-terminal domain (CTD) of RNA Polymerase II (RNAPII). Our preliminary data indicate that HIV-1 Tat may also utilizes CDK2/cyclin E to increase CTD phosphorylation and to stimulate HIV-1 transcription at the G1/S phase of the cell cycle. We hypothesize that, along with CDKg, CDK2 is part of the transcription elongation complex that is required for Tat-dependent transcription. We propose (1) that CDK2/cyclin E binds to the activation domain of Tat and phosphorylates Tat, (2) that this dynamic association of Tat with CDK2/cyclin E along with the interaction of Tat with CTD stimulates phosphorylation of position 2 serines (Ser-2) of the CTD heptapetide repeats, and (3) that this phosphorylation of Ser-2 by CDK2 participates in the regulation of HIV-1 transcription elongation. We also propose that Tat-induced phosphorylation of Ser-2 by CDK2 complements Tat-induced phosphorylation of Ser-5 by CDK9 to stimulate elongation of the HIV-1 transcripts. In the Aim 1 we will determine if CDK2 interacts with Tat in cultured cells and whether this interaction is important for the phosphorylation of Ser-2 of RNAPII heptads during HIV-1 transcription. In the Aim 2 we will determine if CDK2 phosphorylates Tat in cultured cells and whether the phosporylation of Tat is important for HIV-1 transcription. In the Aim 3 we will determine if Tat interacts with cyclin E and whether this interaction regulates HIV-1 transcription. The proposed research will determine whether CDK2/cyclin E along with CDK9/cyclin T1 is a co-activator of Tat-transactivation in vivo. The novelty of the research lies in establishing a previously unrecognized function of Tat as an enzymatic cofactor in HIV-1 transcription, binding simultaneously CDK2, the enzyme, and CTD, the substrate, to facilitate the phosphorylation of CTD Ser-2. The proposed research is important because it may delineate the mechanism of host CDK2 regulation of HIV-1 activated transcription, and may point to host CDK2 as a potential new target for anti-HIV-1 therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI056973-02
Application #
6765864
Study Section
AIDS and Related Research 8 (AARR)
Program Officer
Miller, Roger H
Project Start
2003-07-01
Project End
2007-06-30
Budget Start
2004-07-01
Budget End
2007-06-30
Support Year
2
Fiscal Year
2004
Total Cost
$274,201
Indirect Cost
Name
Howard University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
056282296
City
Washington
State
DC
Country
United States
Zip Code
20059
Nekhai, Sergei; Xu, Min; Foster, Altreisha et al. (2013) Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin. Haematologica 98:455-63
Jerebtsova, Marina; Kumari, Namita; Obuhkov, Yuri et al. (2012) Adenoviral E4 gene stimulates secretion of pigmental epithelium derived factor (PEDF) that maintains long-term survival of human glomerulus-derived endothelial cells. Mol Cell Proteomics 11:1378-88
Ammosova, Tatiana; Obukhov, Yuri; Kotelkin, Alexander et al. (2011) Protein phosphatase-1 activates CDK9 by dephosphorylating Ser175. PLoS One 6:e18985
Debebe, Zufan; Ammosova, Tatyana; Jerebtsova, Marina et al. (2007) Iron chelators ICL670 and 311 inhibit HIV-1 transcription. Virology 367:324-33
Nekhai, Sergei; Jerebtsova, Marina; Jackson, Angela et al. (2007) Regulation of HIV-1 transcription by protein phosphatase 1. Curr HIV Res 5:3-9
Epie, Nicolas; Ammosova, Tatyana; Turner, Willie et al. (2006) Inhibition of PP2A by LIS1 increases HIV-1 gene expression. Retrovirology 3:65
Epie, Nicolas; Ammosova, Tatyana; Sapir, Tamar et al. (2005) HIV-1 Tat interacts with LIS1 protein. Retrovirology 2:6
Ammosova, Tatyana; Jerebtsova, Marina; Beullens, Monique et al. (2005) Nuclear targeting of protein phosphatase-1 by HIV-1 Tat protein. J Biol Chem 280:36364-71
Ammosova, Tatyana; Washington, Kareem; Debebe, Zufan et al. (2005) Dephosphorylation of CDK9 by protein phosphatase 2A and protein phosphatase-1 in Tat-activated HIV-1 transcription. Retrovirology 2:47