Structural determinants of murine leukemia virus reverse transcriptase that affect the frequency of template switching

Retroviral reverse transcriptases (RTs) frequently switch templates within the same RNA or between copackaged viral RNAs to generate mutations acid recombination. To identify structural elements of murine leukemia virus RT important for template switching, we developed an in vivo assay in which BT template switching within direct repeats functionally reconstituted the green fluorescent protein gene. We quantified the effect of mutations in the YXDD motif, the deoxynucleoside triphosphate binding site, the thumb domain, and the RNase II domain of RT and hydroxyurea treatment on the frequencies of template switching. Hydroxyurea treatment and some mutations in RT increased the frequency of RT template switching up to fivefold, while all of the mutations tested in the RNase Il domain decreased the frequency of template switching by twofold. Based on these results, we propose a dynamic copy choice model in which both the rate of DNA polymerization and the rate of RNA degradation influence the frequency of RT template switching.