ONE RETROVIRAL RNA IS SUFFICIENT FOR SYNTHESIS OF VIRAL-DNA

We used previously characterized spleen necrosis virus-based retrovira l vectors and helper cells to study the strand transfers that occur du ring the reverse-transcription phase of a single cycle of retroviral r eplication. The conditions used selected only for formation of an acti ve provirus rather than for expression of multiple drug resistance mar kers. In nonrecombinant proviruses the minus- and plus-strand DNA prim er transfers were almost completely intramolecular. However, as previo usly reported, recombinant proviruses contained approximately equal pr oportions of inter- and intramolecular minus-strand DNA primer transfe rs. Thus, we conclude that in the absence of recombination, one molecu le of retroviral RNA is sufficient for viral DNA synthesis. Large dele tions and deletions with insertions were detected primarily at a limit ed number of positions which appear to be hot spots for such events, t he primer binding site and regions containing multiple inverted repeat s. At these hot spots, the rate of deletions and deletions with insert ions visible with PCR was about 10% per genome per replication cycle. Other deletions and deletions with insertions (detectable,vith PCR) oc curred at a rate of about 0.5%/kb per replication cycle. Crossovers oc curred at a rate of about 6%/kb per replication cycle under single-sel ection conditions. This rate is comparable to the rate that we reporte d previously under double-selection conditions, indicating that retrov iral homologous recombination is not highly error prone. The combined rates of deletions and deletions with insertions at hot spots (10% per genome per replication cycle) and other sites (0.5%/kb per replicatio n cycle) and the rate of crossovers (6%/kb per replication cycle) indi cate that on average, full-size (10-kb) type C retroviruses undergo an additional or aberrant strand transfer about once per cycle of infect ion.