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-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. Nucleoside analogs have been employed to understand the structural basis of PPT recognition in HIV-1 and the LTR-retrotransposon Ty3. These include: (a) thymine and cytosine isosteres, which locally remove hydrogen bonding (Rausch and Le Grice, 2004; Rausch et al., 2003; Lener et al., 2003); (b) locked nucleic acids, which locally decrease flexibility (Dash et al., J. Biol. Chem. 2004); (c) pyrrolo-dC, whose fluorescence is decreased in the double-stranded state (Dash et al., Nucleic Acids Res. 2004); and (d) abasic tetrahydrofuran lesions, which locally remove the base while preserving the sugar-phosphate backbone (Yi-Brunozzi & Le Grice, 2005). Collectively, these studies have highlighted important protein-nucleic acid interactions controlling PPT recognition. NMR studies have been initiated to understand the structural basis for PPT recognition in retroviruses and LTR-retrotransposons, suggesting that sugar pucker may play an important role in these events.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010492-03
Application #
7291836
Study Section
(RML)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2005
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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