Abstract

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.]

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010493-06
Application #
7733055
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2008
Total Cost
$681,864
Indirect Cost

  Institution

Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code

  Publications

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
Williams, Kerry L; Zhang, Yijun; Shkriabai, Nick et al. (2005) Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor. J Biol Chem 280:7949-55
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
Shkriabai, Nick; Patil, Sachindra S; Hess, Sonja et al. (2004) Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry. Proc Natl Acad Sci U S A 101:6894-9
Miller, Jennifer T; Khvorova, Anastasia; Scaringe, Stephen A et al. (2004) Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes. Nucleic Acids Res 32:4687-95
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
Post, Klara; Guo, Jianhui; Howard, Kathryn J et al. (2003) Human immunodeficiency virus type 2 reverse transcriptase activity in model systems that mimic steps in reverse transcription. J Virol 77:7623-34
Roda, Ricardo H; Balakrishnan, Mini; Hanson, Mark N et al. (2003) Role of the Reverse Transcriptase, Nucleocapsid Protein, and Template Structure in the Two-step Transfer Mechanism in Retroviral Recombination. J Biol Chem 278:31536-46

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