Although HIV-1 integrase remains a priority target for development of small molecule antagonists, a high resolution structure of the intact molecule remains elusive. Using a combination of protein footprinting and mass spectrometry, we have been successful in defining the binding site for pyridoxal phosphate on intact HIV-1 integrase. NMR studies with the polypurine tract primer of (+) strand DNA synthesis have demonstrated a change in sugar ring conformation at the PPT-U3 junction, suggesting this may be an important determinant for its recognition by the RNase H domain of HIV-1 RT. Finally, targeted insertion of nucleoside analogs has defined regions the nucleic acid substrate which interact with the DNA polymerase domain of Ty3 RT. An extension of this study investigated whether Ty3 RT mutants were capable of reversing analog-induced inhibition of DNA synthesis. Such biochemical complementation highlighted residues of the Ty3 thumb subdomain that contact individual bases of the template-primer duplex, illustrating the important of nucleoside analog interference strategies. [Corresponds to Le Grice Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program.]

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010493-05
Application #
7592729
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2007
Total Cost
$517,006
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Wendeler, Michaela; Lee, Hsiu-Fang; Bermingham, Alun et al. (2008) Vinylogous ureas as a novel class of inhibitors of reverse transcriptase-associated ribonuclease H activity. ACS Chem Biol 3:635-44
Kvaratskhelia, Mamuka; Grice, Stuart F J Le (2008) Structural analysis of protein-RNA interactions with mass spectrometry. Methods Mol Biol 488:213-9
Bampi, Carole; Bibillo, Arkadiusz; Wendeler, Michaela et al. (2006) Nucleotide excision repair and template-independent addition by HIV-1 reverse transcriptase in the presence of nucleocapsid protein. J Biol Chem 281:11736-43
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Rigourd, Mickael; Bec, Guillaume; Benas, Philippe et al. (2003) Effects of tRNA 3 Lys aminoacylation on the initiation of HIV-1 reverse transcription. Biochimie 85:521-5
Le Grice, Stuart F J (2003) ""In the beginning"": initiation of minus strand DNA synthesis in retroviruses and LTR-containing retrotransposons. Biochemistry 42:14349-55

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