In vitro analysis of the effects of mutations in the G-tract of the human immunodeficiency virus type 1 polypurine tract on RNase H cleavage specificity

The recognition and precise cleavage of the polypurine tract (PPT) of the human immunodeficiency virus type 1 (HIV-1) is an essential step in HIV-1 reverse transcription. The accurate cleavage, and the subsequent removal, of the PPT by the RNase H activity of HIV-1 RT defines the left end of the double-stranded viral DNA genome, the substrate for integration into the host genome. Previous analyses have shown that mutations in the 3′-end (G-tract) of the PPT cause alterations in RNase H cleavage specificity. In particular, mutations at positions 2 and 5 in the G-tract increased the frequency of retention of PPT sequences in the 2-LTR circle junction. To better understand why these mutations affected PPT cleavage in vivo, we analyzed the cleavage of PPT substrates in vitro that contained altered sequences and unusual base substitutions. Our results, herein, confirm that mutations at positions 2 and 5 of the G-tract do significantly alter the cleavage specificity at the PPT/U3 junction, and further suggest that the miscleavages observed in vivo were due to an improper generation of the PPT primer, as opposed to its improper removal. Finally, our results point to the structure of the PPT, rather than the base-specific contacts between the PPT and HIV-1 RT, as the primary determinants of RNase H cleavage specificity at the PPT/U3 junction.