tract primer defines regions controlling initiation of plus-strand DNA synthesis

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 50 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 50 terminus of the (+)-strand primer, whereas the rG:dC tract serves as the prim-ary determinant of initiation specificity. Subsequent analysis demonstrated that G!A or A!G substitu-tion 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 nucleo-side 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 determin-ant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting 4G with an analog containing a 6-NH2 moiety