The RT Biochemistry Section seeks to understand, through protein and nucleic acid mutagenesis, how HIV-1 reverse transcriptase (RT) interacts with the conformationally distinct nucleic acid duplexes encountered while converting the single-stranded RNA genome into an integration-competent double-stranded DNA. Using a combination of methods, we are investigating the synthetic (RNA- and DNA-dependent polymerase) and degradative [ribonuclease H (RNase H)] properties of this multifunctional retroviral enzyme, which continues to be a major target for development of therapeutic agents to inhibit HIV-1 replication and stem the progression of AIDS. (+) Strand DNA synthesis in retroviruses and LTR-containing retrotransposons initiates from the polyrpurine tract (PPT), the precision of which is critical to the integrity of the 5' LTR and its recognition by the viral integration machinery. Although PPT utilization has been studied with respect to alterations in RT or the sequence of the element, the molecular basis underlying this event remains obscure. Two recent studies from the RT Biochemistry Section have shed light on this processing, suggesting that the PPT actively participates in its processing by sequestering RT in an orientation placing the RNase H catalytic center over the biologically relevant processing site. The use of modified nucleosides allows the manner in which RT processes specialized substrates encountered during replication to be examined in detail. We evaluated structural features of the HIV-1 PPT by KMnO4 footprinting and site-directed mutagenesis, and processing of the Saccharomyces cerevisiae retrotransposon Ty3 PPT by targeted introduction of the non-hydrogen-bonding thymine isostere 2,4-difluorotoluene (F). Both studies suggest that structural features of the PPT sequester the polymerizing machinery in an orientation placing its RNase H catalytic center over the biologically relevant cleavage site. Future projects will extend the nucleoside analog strategy to the HIV-1 PPT to include placement of purine analogs in the RNA primer, and non-hydrogen-bonding T and C analogs (F and D, respectively) into the DNA template. Conformationally restricted RNA/DNA hybrids, containing locked nucleic acid (LNA)-inducing nucleosides in the RNA primer, will be investigated. Finally, the structure of the HIV-1 and Ty3 PPTs will be investigated using 19F-NMR and fluorine-substituted nucleoside analogs.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010492-01
Application #
6952084
Study Section
(RML)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Mandal, Dibyakanti; Dash, Chandravanu; Le Grice, Stuart F J et al. (2006) Analysis of HIV-1 replication block due to substitutions at F61 residue of reverse transcriptase reveals additional defects involving the RNase H function. Nucleic Acids Res 34:2853-63
Dash, Chandravanu; Fisher, Timothy S; Prasad, Vinayaka R et al. (2006) Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog interference. J Biol Chem 281:27873-81
Yi-Brunozzi, Hye Young; Brabazon, Danielle M; Lener, Daniela et al. (2005) A ribose sugar conformational switch in the LTR-retrotransposon Ty3 polypurine tract-containing RNA/DNA hybrid. J Am Chem Soc 127:16344-5
Ignatov, Michael E; Berdis, Anthony J; Le Grice, Stuart F J et al. (2005) Attenuation of DNA replication by HIV-1 reverse transcriptase near the central termination sequence. Biochemistry 44:5346-56
Bibillo, Arkadiusz; Lener, Daniela; Tewari, Alok et al. (2005) Interaction of the Ty3 reverse transcriptase thumb subdomain with template-primer. J Biol Chem 280:30282-90
Bibillo, Arkadiusz; Lener, Daniela; Klarmann, George J et al. (2005) Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase. Nucleic Acids Res 33:171-81
Yi-Brunozzi, Hye Young; Le Grice, Stuart F J (2005) Investigating HIV-1 polypurine tract geometry via targeted insertion of abasic lesions in the (-)-DNA template and (+)-RNA primer. J Biol Chem 280:20154-62
Rausch, Jason W; Le Grice, Stuart F J (2004) 'Binding, bending and bonding': polypurine tract-primed initiation of plus-strand DNA synthesis in human immunodeficiency virus. Int J Biochem Cell Biol 36:1752-66
Dash, Chandravanu; Yi-Brunozzi, Hye-Young; Le Grice, Stuart F J (2004) Two modes of HIV-1 polypurine tract cleavage are affected by introducing locked nucleic acid analogs into the (-) DNA template. J Biol Chem 279:37095-102
Dash, Chandravanu; Rausch, Jason W; Le Grice, Stuart F J (2004) Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3' polypurine tract. Nucleic Acids Res 32:1539-47

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