Abstract
In M13mp2 lacZα forward mutation assays measuring intrinsic fidelity of DNA-dependent DNA synthesis, wild-type human immunodeficiency virus type 1 (HIV-1) RTs of group M/subtype B previously showed >10-fold higher error rates than murine leukaemia virus (MLV) and avian myeloblastosis virus (AMV) RTs. An adapted version of the assay was used to obtain error rates of RNA-dependent DNA synthesis for several RTs, including wild-type HIV-1BH10, HIV-1ESP49, AMV and MLV RTs, and the high-fidelity mutants of HIV-1ESP49 RT K65R and K65R/V75I. Our results showed that there were less than two-fold differences in fidelity between the studied RTs with error rates ranging within 2.5 × 10−5 and 3.5 × 10−5. These results were consistent with the existence of a transcriptional inaccuracy threshold, generated by the RNA polymerase while synthesizing the RNA template used in the assay. A modest but consistent reduction of the inaccuracy threshold was achieved by lowering the pH and Mg2+ concentration of the transcription reaction. Despite assay limitations, we conclude that HIV-1BH10 and HIV-1ESP49 RTs are less accurate when copying DNA templates than RNA templates. Analysis of the RNA-dependent mutational spectra revealed a higher tendency to introduce large deletions at the initiation of reverse transcription by all HIV-1 RTs except the double-mutant K65R/V75I.
Highlights
Synthesis for several reverse transcriptase (RT), including wild-type HIV-1BH10, HIV-1ESP49, avian myeloblastosis virus (AMV) and murine leukaemia virus (MLV) RTs, and the highfidelity mutants of HIV-1ESP49 RT K65R and K65R/V75I
RTs play a fundamental role behind these developments, and wild-type (WT) and engineered RT variants of avian myeloblastosis virus (AMV), murine leukaemia virus (MLV), human immunodeficiency virus type 1 (HIV-1) and Geobacillus stearothermophilus group II introns have been developed into more efficient tools to study gene expression by increasing catalytic efficiency, processivity, thermostability or fidelity of DNA synthesis[6,7,8,9]
A commercial T7 RNA polymerase (RNAP) was used to synthesize a lacZα RNA template that was reverse transcribed by retroviral RTs
Summary
Fidelity of RNA-dependent DNA synthesis of retroviral RTs. M13mp[2] lacZα forward mutation assays provide a broad estimate of the fidelity of RTs, based on a relatively large number of mutational sites and sequence contexts, silent mutations cannot be detected by using this method. We determined mutant frequencies and error rates of RNA-dependent DNA synthesis for WT MLV, AMV, HIV-1BH10 and HIV-1ESP49 RTs, as well as mutant RTs O_K65R and O_K65R/V75I For this purpose, a commercial T7 RNA polymerase (RNAP) was used to synthesize a lacZα RNA template that was reverse transcribed by retroviral RTs (the method is outlined in the Supplementary Fig. S1). Reactions were carried out at different pH and Mg2+ concentrations, either in the presence of all four NTPs or with biased NTP pools (i.e. lacking or with a very low concentration of one ribonucleotide) (Supplementary Fig. S8) In this assay, when all NTPs were present (lanes marked with an asterisk), the polymerase synthesized an RNA of 53 nucleotides.
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