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

Abstract

Reverse transcription coupled with DNA amplification has become a wellestablished and powerful molecular technique for studying ribonucleic acids. However, the efficiency of those reactions is largely dependent on the molecular properties of currently used reverse transcriptases (RTs). Engineered and natural RT variants with improved thermostability and fidelity of DNA synthesis should be of great utility in the amplification of RNA targets. In this study, we demonstrate that the wild-type (WT) HIV-1 group O (O_WT) RT shows increased thermostability in comparison with Moloney murine leukemia virus RT and a prototypic HIV-1 group M: subtype B (BH10_WT) RT, while rendering higher yields in reverse transcription PCRs that included a cDNA synthesis step performed at a high temperature range (57–69 °C). In addition, the O_WT RT showed 2.5- fold increased accuracy in M13 lacZα forward mutation assays in comparison with the BH10_WT RT. Unlike the BH10_WT enzyme, O_WT RT showed a very low error rate for frameshifts. Mutational hot spots induced by O_WT RT occurred at nucleotide runs, suggesting a dislocationmediated mechanism for the generation of base substitutions. In HIV-1 group O RT, substituting Ile75 for Val rendered an enzyme that was 1.9 and 4.7 times more faithful than O_WT RT and BH10_WT RTs, respectively, in forward mutation assays. The mutant RT also showed increased misinsertion and mispair extension fidelity in kinetic assays. However, its mutational spectrum was similar to that obtained with the WT group O polymerase. V75I caused a loss of efficiency of reverse transcription PCR amplifications at 65 and 68 °C in comparison with O_WT RT. However, a double mutant devoid of RNase H activity (V75I/E478Q) was found to reverse-transcribe at temperatures as high as 68 °C, while maintaining the increased accuracy of the V75I mutant.