Structural determinants of murine leukemia virus reverse transcriptase that are important for template switching, fidelity, and drug-resistance [electronic resource] /

by Svarovskaia, Evguenia S.

Retroviral reverse transcriptases (RTs) frequently switch templates within the same RNA (intramolecular) or between copackaged viral RNAs (intermolecular). Template switching by RT is necessary for completion of reverse transcription and results in the generation of mutations and recombination, which increase variation in retroviral populations. The structural features of retroviral RTs that affect the frequency of template switching are currently unknown. To identify structural elements of murine leukemia virus (MLV) RT important for template switching, we developed an in vivo assay. This assay used a retroviral vector encoding directly repeated sequences composed of overlapping fragments of the green fluorescent protein gene (GFP) that was permitted to undergo one round of reverse transcription. Template switching within the direct repeats functionally reconstituted GFP. We quantified the effect of mutations in RT on the frequencies of template switching. Using MLV RTs containing single amino acid substitution mutations in the Tyr-X-Asp-Asp (YXDD) motif, the dNTP binding site, the thumb domain, and the RNase H domain were characterized. The results showed that some mutations in the YXDD motif and dNTP binding site increased the frequency of template switching, while all mutations in the RNase H domain tested decreased the same frequency. Hydroxyurea treatment, which depleted nucleotide pools and reduced the rate of DNA synthesis, increased the frequency of template switching for the wild-type RT and the YXDD mutant RTs, but not for the RNase H mutant RTs. Based on these results, we proposed a dynamic copy- 35 choice model in which both the rate of DNA polymerization and the rate of degradation of the RNA template influence the frequency of RT template switching.