Specificity of initiation of plus-strand DNA by Rous sarcoma virus

We previously reported that in the endogenous reaction of Rous sarcoma virus disrupted by melittin, plus-strand DNA initiates on a small oligonucleotide primer and that this initiation can be reconstructed in vitro in reactions containing purified minus-strand DNA as template, viral RNA as a source of primer, and reverse transcriptase (Smith et al., J. Virol. 49:200-204, 1984). Further studies on the specificity of initiation in the endogenous reaction have shown the following. (i) The primer was 12 nucleotides in length. Its sequence began with a 5' pyrimidine, followed by 11 purines, ending with rGrA-3'. This sequence was in agreement with the known plus-strand RNA sequence immediately upstream from the initiation site. Thus, the primer began one nucleotide 5' to the so-called polypurine tract that has been found on all retrovirus genomes. (ii) The transition point between RNA primer and DNA product was precisely located. It was before the end of the polypurine tract. Thus the polypurine tract, although essential for virus replication and probably a flag for the priming event, did not define the limits of the RNA primer. After primer removal, the DNA had a 5' phosphate, consistent with generation by the viral RNase H activity. The priming specificity in reconstructed reactions was also examined further, with the following observations. (i) When the source of RNA primer was prehybridized to the template viral DNA, the generation, utilization, and subsequent removal of primer were essentially the same as those observed in the endogenous reaction. In the absence of deliberate prehybridization, some specificity was lost. There were than additional locations for the 5' end of the primer as well as the transition point between RNA primer and DNA. (ii) Purine-rich oligoribonucleotides created by RNase A digestion of viral RNA could prime strong-stop plus DNA, but again with the loss of specificity relative to that in the endogenous reaction. (iii) The 5' end of the minus-strand DNA template was not required for initiation of strong-stop plus DNA. Therefore, the specificity of initiation did not depend upon an intramolecular interaction requiring the two inverted repeat sequences that flank the long terminal repeat.