This proposal describes single molecule kinetic and dynamic investigations of critical protein/nucleic acid intermediates in the reverse transcription mechanism of HIV-1. HIV-1 utilizes the nucleocapsid protein (NC), a nucleic acid chaperone, to facilitate a variety of steps critical to the reverse transcription of its RNA genome. In the first step of reverse transcription, HIV-1 uses tRNA(Lys,3) to prime minus strand DNA synthesis. NC has been shown to induce the unwinding of tRNA(Lys,3) and to facilitate its annealing to the RNA genome. NC again catalyzes nucleic acid rearrangements in two subsequent strand transfer steps critical to reverse transcription. The molecular-level picture of how NC executes its nucleic acid chaperone functions is unclear and will be elucidated by the fluorescence single molecule spectroscopy (SMS) methods in the proposed work, including fluorescence resonance energy transfer studies on various immobilized dyelabeled NC, RNA, and DNA model compounds and their complexes. In particular, SMS will be used to determine the kinetics and mechanisms of NC-induced unwinding and annealing of base-paired DNAs/RNAs to the HIV-1 RNA genome. The sequence of molecular rearrangements, the rate-limiting kinetic steps, and the molecularity of important reaction intermediates will be revealed using SMS methods. More generally, this work strives to develop powerful new strategies for molecular-level biochemical investigations of HIV-1 and related systems with highly heterogeneous kinetics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065818-04
Application #
7059910
Study Section
Special Emphasis Panel (ZRG1-AARR-1 (01))
Program Officer
Lewis, Catherine D
Project Start
2003-05-01
Project End
2008-04-30
Budget Start
2006-05-01
Budget End
2008-04-30
Support Year
4
Fiscal Year
2006
Total Cost
$241,465
Indirect Cost
Name
University of Texas Austin
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78712
Zeng, Yining; Liu, Hsiao-Wei; Landes, Christy F et al. (2007) Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer. Proc Natl Acad Sci U S A 104:12651-6
Landes, Christy F; Zeng, Yining; Liu, Hsiao-Wei et al. (2007) Single-molecule study of the inhibition of HIV-1 transactivation response region DNA/DNA annealing by argininamide. J Am Chem Soc 129:10181-8
Liu, Hsiao-Wei; Zeng, Yining; Landes, Christy F et al. (2007) Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription. Proc Natl Acad Sci U S A 104:5261-7
Cosa, Gonzalo; Zeng, Yining; Liu, Hsiao-Wei et al. (2006) Evidence for non-two-state kinetics in the nucleocapsid protein chaperoned opening of DNA hairpins. J Phys Chem B 110:2419-26
Liu, Hsiao-Wei; Cosa, Gonzalo; Landes, Christy F et al. (2005) Single-molecule FRET studies of important intermediates in the nucleocapsid-protein-chaperoned minus-strand transfer step in HIV-1 reverse transcription. Biophys J 89:3470-9
Cosa, Gonzalo; Harbron, Elizabeth J; Zeng, Yining et al. (2004) Secondary structure and secondary structure dynamics of DNA hairpins complexed with HIV-1 NC protein. Biophys J 87:2759-67