EXTENDED MINUS-STRAND DNA AS TEMPLATE FOR R-U5-MEDIATED 2ND-STRAND TRANSFER IN RECOMBINATIONAL RESCUE OF PRIMER BINDING SITE-MODIFIED RETROVIRAL VECTORS

We have previously demonstrated recombinational rescue of primer bindi ng site (PBS)-impaired Akv murine leukemia virus-based vectors involvi ng initial priming on endogenous viral sequences and template switchin g during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen ct al., J. Viral. 70:143 9-1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on en dogenous virus RNA, read-through of the mutated PBS during minus-stran d synthesis, and subsequent second-strand transfer mediated by the R-U 5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms far R-U5-mediated second-strand transfe r and its possible role in retrovirus replication and evolution are di scussed.