Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors

We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 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 endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.

我们此前已证实，针对引物结合位点（primer binding site, PBS）受损的Akv鼠白血病病毒载体，可通过重组拯救技术实现救援：该过程依赖于内源性病毒序列上的初始引发，以及cDNA合成期间的模板转换，从而在逆转录的第二链转移阶段获得PBS互补性（Mikkelsen等，《病毒学杂志》（J. Virol.），1996年，70卷：1439–1447）。借助同一强制重组系统，我们此次发现了结构各异的重组前病毒，这表明PBS敲除载体可通过以下途径实现救援：首先在内源性病毒RNA上启动初始引发，随后在负链合成过程中通读突变的PBS，最终通过正链与延伸后的负链DNA受体模板之间的R-U5互补性介导后续的第二链转移。本文还探讨了R-U5介导的第二链转移机制，及其在逆转录病毒复制与进化中的潜在作用。