Single-molecule spectroscopy studies of several nucleoprotein complexes demonstrated that HIV-1 reverse transcriptase (RT) assumes different orientations, depending on the nature of the nucleic acid substrate. An unexpected outcome was our observation that nonnucleoside RT inhibitors (NNRTIs) induce enzyme binding to the polypurine tract (PPT) in an orientation incompatible with initiating DNA synthesis. Subsequent studies examined tRNA-primed initiation of (-) strand DNA synthesis, showing that the base-paired U5-IR stem immediately downstream of the primer binding site controls enzyme orientation and that this barrier to initiation can be alleviated by including the HIV-1 nucleocapsid (NC) protein. Single-molecule spectroscopy will be continued to examine the effect of NC-containing Gag precursors and RT mutations on (a) tRNA-primed initiation on the HIV-1 genome, (b) tRNALys,3-primed initiation on related lentiviral RNAs, (c) dynamics of termination at the central termination sequence (CTS), and (d) conformational dynamics of a gammaretroviral RT. These projects will be combined with new strategies for site-specific placement of fluorophores within RT subunits containing AZ-Phe via Click chemistry.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Ganji, Mahipal; Docter, Margreet; Le Grice, Stuart F J et al. (2016) DNA binding proteins explore multiple local configurations during docking via rapid rebinding. Nucleic Acids Res 44:8376-84
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
Chung, Suhman; Miller, Jennifer T; Lapkouski, Mikalai et al. (2013) Examining the role of the HIV-1 reverse transcriptase p51 subunit in positioning and hydrolysis of RNA/DNA hybrids. J Biol Chem 288:16177-84
Le Grice, Stuart F J (2012) Human immunodeficiency virus reverse transcriptase: 25 years of research, drug discovery, and promise. J Biol Chem 287:40850-7