Wild-type HIV-1 group O RT (reverse transcriptase) shows increased thermostability in comparison with HIV-1 group M subtype B RT and MLV (murine leukaemia virus) RT. However, its utility in the amplification of RNA targets is limited by the reduced accuracy of lentiviral RTs compared with oncoretroviral RTs (i.e. MLV RT). The effects of the mutations K65R, R78A and K65R/V75I on the fidelity of HIV-1 group O RTs were studied using gel-based and M13mp2 lacZ forward-mutation fidelity assays. Forward-mutation assays demonstrated that mutant RTs K65R, R78A and K65R/V75I showed >9-fold increased accuracy in comparison with the wild-type enzyme and were approximately two times more faithful than the MLV RT. Compared with MLV RT, all of the tested HIV-1 group O RT variants showed decreased frameshift fidelity. However, K65R RT showed a higher tendency to introduce one-nucleotide deletions in comparison with other HIV-1 group O RT variants. R78A had a destabilizing effect on the RT, either in the presence or absence of V75I. At temperatures above 52 °C, K65R and K65R/V75I retained similar levels of DNA polymerase activity to the wild-type HIV-1 group O RT, but were more efficient than HIV-1 group M subtype B and MLV RTs. K65R, K65R/V75I and R78A RTs showed decreased misinsertion and mispair extension fidelity in comparison with the wild-type enzyme for most base pairs studied. These assays revealed that nucleotide selection is mainly governed by kpol (pol is polymerization) in the case of K65R, whereas both kpol and Kd affect nucleotide discrimination in the case of K65R/V75I.

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.