Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases

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.

Translesion synthesis by AMV, HIV, and MMLVreverse transcriptases using RNA templates containing inosine, guanosine, and their 8-oxo-7,8-dihydropurine derivatives

“…Since it is unlikely that the protonation states of the nucleobases change in this range, the most probable cause for this observation may be due to varying interactions between the enzyme:duplex:dNTP complex. Importantly this trend has been previously reported, where decreasing the pH leads to increased fidelity while increasing the pH leads to the opposite result [52].…”

“…On the other hand, efficient cDNA synthesis proceeded in the case where a canonical G:C base pair was formed (duplex 1:7, lane 6). Experiments carried out on duplexes 1:8-4:8 (Fig 7, bottom-containing a dT opposite the modification) also led to efficient addition of dTTP (and cDNA synthesis) when using templates containing G, I, or 8-oxoI (52,54,64,66,70,72), with 8-oxoG as the exception (lanes 58, 60). Lastly to explore the impact of a modified base pair two positions away from the transcription site, experiments were carried out using duplexes 1:10-4:10 and 1:11-4:11.…”

The Determination of HIV-1 RT Mutation Rate, Its Possible Allosteric Effects, and Its Implications on Drug Resistance

“…For a better study of HIV-1 RT, the mutation positions are important to provide mechanistic insights to the development of drug resistance [76]. The well-established mutation rate of HIV-1 RT is performed on lacZα [39][40][41][42][43][44][45]48,58,77], resulting in a gap of knowledge regarding how HIV-1 genes are mutated by RT.…”

The Determination of HIV-1 RT Mutation Rate, Its Possible Allosteric Effects, and Its Implications on Drug Resistance

“…When considering the mutation rates of HIV-1 RT and the emergence on HIV, the locations where these mutations occur are important to understand emerging drug resistance [76] as well as potential understanding to how viruses can jump species [77,78]. Although the mutation rate of HIV-1 RT has been widely studied, majority of the studies are performed using the reporter gene lacZα as its template [39][40][41][42][43][44][45]48,58,79], resulting in a major gap of understanding on how HIV-1 RT mutates HIV-1 genes directly.…”