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 importance of nucleoside analog interference strategies. Selective 2'hydroxyl acylation analyzed by primer extension (SHAPE) examines RNA secondary structure via sensitivity of the ribose 2'OH in an unpaired configuration to acylation. Our initial SHAPE studies focused on wild-type and mutant HIV-1 Rev response elements (RREs) and subsequently examined the minimal transport element of the murine LTR-retrotransposon MusD. In parallel, we expanded this technology by developing methods to examine the structure of short RNA/DNA hybrids by mass spectrometry (SHAMS) and defining tertiary interactions via antisense-interfered SHAPE (ai-SHAPE). While structural analysis of regulatory RNAs will continue, we recognize that SHAPE is not a stand-alone technique. Chemo-enzymatic footprinting will therefore be complemented with (a) NMR spectroscopy, where the size of the RNA permits, (b) small-angle X-ray scattering, and (c) targeted Fe-EDTA-footprinting with """"""""threading intercalators,"""""""" prepared in collaboration with the CCR Chemical Biology Laboratory. A long-term goal of our Section is to examine RNA structure in the context of the viral RNA genome, and novel approaches to increase SHAPE sensitivity for in vivo footprinting will be investigated. [Corresponds to Le Grice Project 1 in the October 2011 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 #
1ZIABC010493-11
Application #
8763117
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2013
Total Cost
$689,338
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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
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
Nowak, Elżbieta; 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
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
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 20:230-6
Lusvarghi, Sabrina; Sztuba-Solinska, Joanna; Purzycka, Katarzyna J et al. (2013) RNA secondary structure prediction using high-throughput SHAPE. J Vis Exp :e50243
Huang, Qing; Purzycka, Katarzyna J; Lusvarghi, Sabrina et al. (2013) Retrotransposon Ty1 RNA contains a 5'-terminal long-range pseudoknot required for efficient reverse transcription. RNA 19:320-32
Sztuba-Solinska, Joanna; Teramoto, Tadahisa; Rausch, Jason W et al. (2013) Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome. Nucleic Acids Res 41:5075-89

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