The AIDS virus, HIV, utilizes the host cell factor P-TEFb to regulate its transcriptional and replication program. A small viral protein, Tat, and the 5'untranslated region (UTR) of the viral transcript are both involved in this process. Tat is an RNA binding protein that binds to the trans-activation response element, TAR, that is present in the 5'UTR. Tat can form a complex with TAR and P-TEFb. P-TEFb is a cyclin dependent kinase that controls the elongation phase of transcription, by regulating the transition into productive elongation. P-TEFb itself is regulated by the reversible association of a cellular RNA binding protein HEXIM1 or HEXIM2 and the small nuclear RNA, 7SK. This large form P-TEFb is relatively inactive compared to the free form of P-TEFb, but represents the major form of P-TEFb in many cell types. Therefore, most of the potential P-TEFb activity is held in this large complex. The original model for Tat transactivation emphasized the recruitment of P-TEFb to the nascent viral transcript;however, data recently obtained suggest that HIV utilizes the cellular P-TEFb control machinery more extensively than previously imagined. We plan to examine these broader regulatory mechanisms using a combination of biochemical and structural biological techniques. In this collaborative effort between a biochemistry lab and a structural biology lab, existing in vitro assays will be used to find conditions to generate relevant complexes containing proteins, or proteins and RNA that will form crystals. Finally the structures of these complexes will be solved using crystallographic techniques. The results from these studies will ultimately yield structural and functional information needed for the rational design of therapies targeting HIV, and will provide a basis for further understanding control of cellular transcription by P-TEFb. This project is aimed at understanding how HIV utilizes cellular P-TEFb regulatory machinery to control its own transcriptional program with the prospect of developing rational therapies for HIV. Complexes comprised of viral and cellular proteins and RNAs will be crystallized and structures determined.

Public Health Relevance

This project is aimed at understanding how HIV utilizes cellular P-TEFb regulatory machinery to control its own transcriptional program with the prospect of developing rational therapies for HIV. Complexes comprised of viral and cellular proteins and RNAs will be crystallized and structures determined.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
5R33AI074392-04
Application #
7923882
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Salzwedel, Karl D
Project Start
2007-07-15
Project End
2012-08-31
Budget Start
2010-09-01
Budget End
2011-08-31
Support Year
4
Fiscal Year
2010
Total Cost
$367,983
Indirect Cost
Name
University of Iowa
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Gu, Jianyou; Babayeva, Nigar D; Suwa, Yoshiaki et al. (2014) Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4. Cell Cycle 13:1788-97
Van Duyne, Rachel; Guendel, Irene; Jaworski, Elizabeth et al. (2013) Effect of mimetic CDK9 inhibitors on HIV-1-activated transcription. J Mol Biol 425:812-29
Zhou, Qiang; Li, Tiandao; Price, David H (2012) RNA polymerase II elongation control. Annu Rev Biochem 81:119-43
Cheng, Bo; Li, Tiandao; Rahl, Peter B et al. (2012) Functional association of Gdown1 with RNA polymerase II poised on human genes. Mol Cell 45:38-50
Peterlin, B Matija; Brogie, John E; Price, David H (2012) 7SK snRNA: a noncoding RNA that plays a major role in regulating eukaryotic transcription. Wiley Interdiscip Rev RNA 3:92-103
Fujinaga, Koh; Barboric, Matjaz; Li, Qintong et al. (2012) PKC phosphorylates HEXIM1 and regulates P-TEFb activity. Nucleic Acids Res 40:9160-70
Cojocaru, Marilena; Bouchard, Annie; Cloutier, Philippe et al. (2011) Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B. J Biol Chem 286:5012-22
Tahirov, Tahir H; Babayeva, Nigar D; Varzavand, Katayoun et al. (2010) Crystal structure of HIV-1 Tat complexed with human P-TEFb. Nature 465:747-51
Price, David H (2010) Regulation of RNA polymerase II elongation by c-Myc. Cell 141:399-400
Krueger, Brian J; Varzavand, Katayoun; Cooper, Jeffrey J et al. (2010) The mechanism of release of P-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activators includes a conformational change in 7SK. PLoS One 5:e12335

Showing the most recent 10 out of 11 publications