Increased thermostability and fidelity of DNA synthesis of wild-type and mutant HIV-1 group O reverse transcriptases.

摘要

Reverse transcription coupled with DNA amplification has become a wellestablished\udand powerful molecular technique for studying ribonucleic\udacids. However, the efficiency of those reactions is largely dependent on the\udmolecular properties of currently used reverse transcriptases (RTs).\udEngineered and natural RT variants with improved thermostability and\udfidelity of DNA synthesis should be of great utility in the amplification of\udRNA targets. In this study, we demonstrate that the wild-type (WT) HIV-1\udgroup O (O_WT) RT shows increased thermostability in comparison with\udMoloney murine leukemia virus RT and a prototypic HIV-1 group M:\udsubtype B (BH10_WT) RT, while rendering higher yields in reverse\udtranscription PCRs that included a cDNA synthesis step performed at a\udhigh temperature range (57–69 °C). In addition, the O_WT RT showed 2.5-\udfold increased accuracy in M13 lacZα forward mutation assays in\udcomparison with the BH10_WT RT. Unlike the BH10_WT enzyme, O_WT\udRT showed a very low error rate for frameshifts. Mutational hot spots\udinduced by O_WT RT occurred at nucleotide runs, suggesting a dislocationmediated\udmechanism for the generation of base substitutions. In HIV-1\udgroup O RT, substituting Ile75 for Val rendered an enzyme that was 1.9 and\ud4.7 times more faithful than O_WT RT and BH10_WT RTs, respectively, in\udforward mutation assays. The mutant RT also showed increased misinsertion\udand mispair extension fidelity in kinetic assays. However, its\udmutational spectrum was similar to that obtained with the WT group O\udpolymerase. V75I caused a loss of efficiency of reverse transcription PCR\udamplifications at 65 and 68 °C in comparison with O_WT RT. However, a\uddouble mutant devoid of RNase H activity (V75I/E478Q) was found to\udreverse-transcribe at temperatures as high as 68 °C, while maintaining the\udincreased accuracy of the V75I mutant.