Analysis of plus-strand primer selection, removal, and reutilization by retroviral reverse transcriptases

The ability of reverse transcriptase to generate, extend, and remove the pr imer derived from the polypurine tract (PPT) is vital for reverse transcrip tion, since this process determines one of the ends required for integratio n of the viral DNA. Based on the ability of the RNase H activity of Moloney murine leukemia virus reverse transcriptase to cleave a long RNA/DNA hybri d containing the PPT, it appears that cleavages that could generate the plu s-strand primer can occur by an internal cleavage mechanism without any pos itioning by an RNA 5'-end, and such cleavages may serve to minimize cleavag e events within the PPT itself. If the PPT were to be cleaved inappropriate ly just upstream of the normal plus-strand origin site, the resulting 3'-en ds would not be extended by reverse transcriptase. Extension of the PPT pri mer by at least 2 nucleotides is sufficient for recognition and correct cle avage by RNase H at the RNA-DNA junction to remove the primer. Specific rem oval of the PPT primer after polymerase extension deviates from the general observation that primer removal occurs by cleavage one nucleotide away fro m the RNA-DNA junction and suggests that the same PPT specificity determina nts responsible for generation of the PPT primer also direct PPT primer rem oval. Once the PPT primer has been extended and removed from the nascent pl us-strand DNA, reinitiation at the resulting plus-strand primer terminus do es not occur, providing a mechanism to prevent the repeated initiation of p lus strands.