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Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

  1. Author:
    Post, K.
    Kankia, B.
    Gopalakrishnan, S.
    Yang, V.
    Cramer, E.
    Saladores, P.
    Gorelick, R. J.
    Guo, J. H.
    Musier-Forsyth, K.
    Levin, J. G.

  2. Author Address

    Post, Klara, Gopalakrishnan, Swathi, Yang, Victoria, Cramer, Elizabeth, Saladores, Pilar, Guo, Jianhui, Levin, Judith G.] Eunice Kennedy Shriver Natl Inst Child Hlth & Hum, NIH, Mol Genet Lab, Bethesda, MD 20892 USA. [Kankia, Besik, Musier-Forsyth, Karin] Ohio State Univ, Dept Chem, Columbus, OH 43210 USA. [Kankia, Besik, Musier-Forsyth, Karin] Ohio State Univ, Dept Biochem, Columbus, OH 43210 USA. [Gorelick, Robert J.] SAIC Frederick Inc, NCI, AIDS & Canc Virus Program, Frederick, MD 21702 USA. [Guo, Jianhui] Shanghai Allist Pharmaceut, Shanghai 201203, Peoples R China.
    1. Year: 2009
  2. Journal: Nucleic Acids Research
    1. 37
    2. 6
    3. Pages: 1755-1766
  4. Type of Article: Article
  1. Abstract:

    During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3 polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NCs observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

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  2. DOI: 10.1093/nar/gkn1045
  3. PMID: 19158189

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