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 reverse transcriptase (RT). Finally, targeted insertion of nucleoside analogs has defined regions of the nucleic acid substrate that 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
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010493-09
Application #
8349025
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2011
Total Cost
$789,799
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Sztuba-Solinska, Joanna; Rausch, Jason W; Smith, Rodman et al. (2017) Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins. Nucleic Acids Res 45:6805-6821
Shapiro, Bruce A; Le Grice, Stuart F J (2016) Advances in RNA structure determination. Methods 103:1-3
Rausch, Jason W; Le Grice, Stuart F J (2015) HIV Rev Assembly on the Rev Response Element (RRE): A Structural Perspective. Viruses 7:3053-75
Sherpa, Chringma; Rausch, Jason W; Le Grice, Stuart F J et al. (2015) The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication. Nucleic Acids Res 43:4676-86
Nowak, El?bieta; Miller, Jennifer T; Bona, Marion K et al. (2014) Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry. Nat Struct Mol Biol 21:389-96
Abbondanzieri, Elio A; Le Grice, Stuart F J (2014) Unraveling the gymnastics of reverse transcription through single molecule spectroscopy. AIDS Res Hum Retroviruses 30:209-10
Lapkouski, Mikalai; Tian, Lan; Miller, Jennifer T et al. (2013) Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation. Nat Struct Mol Biol 20:230-236
Lusvarghi, Sabrina; Sztuba-Solinska, Joanna; Purzycka, Katarzyna J et al. (2013) The HIV-2 Rev-response element: determining secondary structure and defining folding intermediates. Nucleic Acids Res 41:6637-49
Chamanian, Mastooreh; Purzycka, Katarzyna J; Wille, Paul T et al. (2013) A cis-acting element in retroviral genomic RNA links Gag-Pol ribosomal frameshifting to selective viral RNA encapsidation. Cell Host Microbe 13:181-92
Lusvarghi, Sabrina; Sztuba-Solinska, Joanna; Purzycka, Katarzyna J et al. (2013) RNA secondary structure prediction using high-throughput SHAPE. J Vis Exp :e50243

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