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Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis

  1. Author:
    Rausch, J. W.
    Le Grice, S. F. J.

  2. Author Address

    NCI, RT Biochem Sect, Resistance Mechanisms Lab, HIV Drug Resistance Program, Frederick, MD 21702 USA.;Le Grice, SFJ, NCI, RT Biochem Sect, Resistance Mechanisms Lab, HIV Drug Resistance Program, Frederick, MD 21702 USA.;slegrice@ncifcrf.gov
    1. Year: 2007
    2. Date: Jan
  2. Journal: Nucleic Acids Research
    1. 35
    2. 1
    3. Pages: 256-268
  4. Type of Article: Article
  5. ISSN: 0305-1048
  1. Abstract:

    Despite extensive study, the mechanism by which retroviral reverse transciptases (RTs) specifically utilize polypurine tract (PPT) RNA for initiation of plus-strand DNA synthesis remains unclear. Three sequence motifs within or adjacent to the purine-rich elements are highly conserved, namely, a rU:dA tract region immediately 5' to the PPT, an rA:dT-rich sequence constituting the upstream portion of the PPT and a downstream rG:dC tract. Using an in vitro HIV-1 model system, we determined that the former two elements define the 5' terminus of the (+)-strand primer, whereas the rG:dC tract serves as the primary determinant of initiation specificity. Subsequent analysis demonstrated that G -> A or A -> G substitution at PPT positions -2, -4 and +1 (relative to the scissile phosphate) substantially reduces (+)-strand priming. We explored this observation further using PPT substrates substituted with a variety of nucleoside analogs [inosine (I), purine riboside (PR), 2-aminopurine (2-AP), 2,6-diaminopurine (2,6-DAP), isoguanine (iG)], or one of the naturally occurring bases at these positions. Our results demonstrate that for PPT positions -2 or +1, substituting position 2 of the purine was an important determinant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting -4G with an analog containing a 6-NH2 moiety.

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  2. DOI: 10.1093/nar/gkl909
  3. View record in Web of Science®
  4. WOS: 000243805800032

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