Abstract
During reverse transcription, the RNase H activity of reverse transcriptase specifically cleaves the viral genome within the polypurine tract (PPT) to create the primer used for the initiation of plus-strand DNA synthesis and nonspecifically cleaves the viral genome to facilitate synthesis of plus-strand DNA. To understand how primer length and sequence affect generation and utilization of the PPT, we employed short hybrid substrates containing or lacking the PPT to evaluate cleavage, extension, and binding by reverse transcriptase. Substrates containing RNAs with the correct 3' end for initiation of plus-strand synthesis were extended equally well by reverse transcriptase, but primer length affected susceptibility to RNase H cleavage. RNA substrates with 3' ends extending beyond the plus-strand initiation site were extended poorly but were specifically cleaved to generate the correct 3' end for initiation of plus-strand synthesis. Substrates containing RNAs lacking the PPT were cleaved nonspecifically and extended inefficiently. Specific cleavages to generate the plus-strand primer and 5'-end-directed cleavages were kinetically favored over cleavages that destroyed the PPT primer or degraded other short RNA fragments. The PPT was not intrinsically resistant to cleavage by the isolated RNase H domain, and the isolated polymerase domain extended RNA primers containing the PPT sequence irrespective of the primer 3' end. These results provide insights into how reverse transcriptase generates and selectively utilizes the PPT primer for initiation of plus-strand DNA synthesis.
Highlights
Moloney murine leukemia virus (M-MuLV)1 converts its single-stranded plus-sense RNA genome into a double-stranded DNA molecule through the replicative process termed reverse transcription
The polymerase-independent activity of RNase H is positioned by the polymerase domain of reverse transcriptase binding at the 5Ј end of an RNA annealed to a longer DNA (23, 28, 30 –32), suggesting that this type of RNase H activity plays a major role in degrading the RNA genome following minus-strand synthesis
Cleavage of Hybrid Oligonucleotide Substrates by RNase H Activity of Reverse Transcriptase—To create the polypurine tract (PPT) primer used for plus-strand DNA synthesis, the RNase H activity of reverse transcriptase cleaves the viral RNA hybridized to minus-strand DNA within the highly conserved PPT sequence between a G nucleotide and an A nucleotide (Fig. 1) [1]
Summary
M-MuLV, Moloney murine leukemia virus; RT, reverse transcriptase; HϪRT, the RNase H-deficient version of reverse transcriptase containing point mutations that eliminate RNase H activity; RT⌬H, a version of reverse transcriptase that completely lacks the RNase H domain; RT⌬Pol, a version of reverse transcriptase that completely lacks the polymerase domain; PPT, polypurine tract; HIV, human immunodeficiency virus; nt, nucleotide(s); DTT, dithiothreitol; BSA, bovine serum albumin; LTRs, long terminal repeats. As minus-sense DNA synthesis progresses, a purine-rich sequence in the RNA genome termed the polypurine tract (PPT) that lies immediately adjacent to a downstream unique sequence (U3) is copied and subsequently cleaved to generate the PPT primer This primer is used to initiate plus-strand DNA synthesis, which extends through U3-R-U5 and continues after a second jump to the 5Ј end of the DNA template. Nealed to longer DNAs to generate model hybrid substrates representing various primer-template possibilities that might occur at or near the beginning of plus-strand DNA synthesis When these substrates were used to assay the polymeraseindependent activity of RNase H, the sequence and length of the RNA dictated the cleavage pattern. These studies offer insights into how reverse transcriptase associates with sequences containing or lacking the PPT and extend our understanding of the initiation of plus-strand DNA synthesis
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