Two in vitro approaches were used to investigate the priming of strong-stop plus DNA by Rous sarcoma virus. This 340-base DNA species is the first major plus-strand DNA product seen in both infected cells and in endogenous reactions of disrupted virions. In the first approach, we set up a reconstructed system in which strong-stop plus DNA was synthesized by reverse transcriptase from a high-molecular-weight minus-strand viral DNA template. This synthesis was shown to be strictly dependent on the addition of primers to the reaction mixture. The addition of high-molecular-weight RNA from both viral and cellular sources, as well as oligodeoxyguanylate, gave specific synthesis of strong-stop plus DNA, whereas the addition of oligodeoxycytidylate-oligodeoxyadenylate and viral 4S RNA did not. In the second approach, strong-stop plus DNA synthesized in melittin-permeabilized virions was examined on a high-resolution polyacrylamide gel. This DNA was shown to have ca. 11 to 13 ribonucleotides at its 5' end. These results indicate that strong-stop plus DNA is initiated on a preformed RNA primer.

We previously reported that in the endogenous reaction of Rous sarcoma virus disrupted by melittin, plusstrand 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 then 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.

The unintegrated linear DNA synthesized in cells infected by Rous sarcoma virus is a predominantly double-stranded structure in which most of the minus-strand DNA, complementary to the viral RNA genome, is genome sized, whereas the plus-strand DNA is present as subgenomic fragments. We previously reported the application of benzoylated naphthoylated DEAE-cellulose chromatography to demonstrate that of the linear viral DNA species synthesized in quail embryo fibroblasts infected with Rous sarcoma virus greater than 99.5% contain single-stranded regions and these regions are predominantly composed of plus-strand DNA sequences (T. W. Hsu and J. M. Taylor, J. Virol. 44:47-53, 1982). We now present the following additional findings. (i) There were on the average 3.5 single-stranded regions per linear viral DNA, and these single-stranded regions could occur at many locations. (ii) With a probe to the long terminal repeat, we detected, in addition to a heterogeneous size distribution of subgenomic plus-strand DNA species, at least three prominent discrete size classes. Each of these discrete species had its own specific initiation site, but all had the same termination site. Such species were analogous to those reported by Kung et al. (J. Virol. 37: 127-138, 1981). (iii) These discrete size classes of plus-strand DNA were present not only on the major size class of linear DNA but also on a heterogeneous of slower-sedimenting species, which we have called immature linears. Our interpretation is that we have thus detected several additional sites for the initiation of plus-strand DNA. (iv) The 340-base plus-strand strong-stop DNA was only found associated with the immature linears. (v) From a size and hybridization comparison of these discrete size classes of plus-strand DNA with minus-strand DNA species, as synthesized in the endogenous reaction of melittin-disrupted virions, it was found that the putative additional initiation sites for plus-strand DNA synthesis corresponded to many of the pause sites in the synthesis of minus-strand DNA.

No abstract

Linial et al. isolated a quail cell line, SE21Q1b, that is transformed by a single integrated provirus of Rous sarcoma virus. Virus particles are released from these cells, but because of a provirus defect, cellular rather than viral RNA is packaged. When these virus particles are disrupted with melittin in the presence of an appropriate reaction mixture containing actinomycin D, there is significant reverse transcription of packaged cellular RNA species. We have shown that (i) cellular 7S L RNA is an efficient template; (ii) initiation is on a unique tRNA-like primer; (iii) synthesis produces a 135-base strong-stop DNA product; and (iv) after synthesis, RNase H acts to remove the 135 bases of the 7S L RNA which acted as the template. A possible facilitator of such specific transcription may be that, in the virus particles but not in the cell, the majority of the 7S L RNA species exist complexed with the tRNA, even before the disruption of the virus. From the size and sequence features of the reverse transcript of 7S L RNA, we speculate that such events may have participated in the process by which animal cell genomes have, in the course of evolution, accumulated multiple copies of Alu-like elements. 278 on July 6, 2020 by guest