Fidelity of human immunodeficiency viruses type 1 and type 2 reverse transcriptases in DNA synthesis reactions using DNA and RNA templates

Resum

Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2, respectively) are the causative agent of acquired immunodeficiency syndrome (AIDS). Most of the available clinically approved antiretroviral drugs directed against HIV inhibit the viral reverse transcriptase (RT), a multifunctional enzyme with RNA- and DNA-dependent DNA polymerase activity, as well as ribonuclease H, strand-transfer, and strand displacement activities. Nucleoside RT inhibitors (NRTIs) constitute the backbone of current therapies against both types of HIV, although the acquisition of NRTI resistance is mediated by the development of different mutational pathways. In HIV-2, resistance to all approved nucleoside analogues is conferred by the combination of RT substitutions K65R, Q151M and M184V. Interestingly, in highly divergent HIV-1 RTs, K65R alone confers >8-fold increased accuracy of DNA synthesis. In this Thesis, we have determined the intrinsic fidelity of DNA synthesis of wild-type (WT) HIV-2 group A (strain ROD) RT and mutants K65R and K65R/Q151M/M184V. Our results show that in HIV-2ROD RT those changes have a relatively small impact on nucleotide selectivity. Furthermore, we found that there were less than 2- fold differences in error rates obtained with forward mutation assays using mutant and WT HIV- 2ROD RTs. A different conformation of the β3-β4 hairpin loop in HIV-1 and HIV-2 RTs could probably explain the different effects of K65R. In addition to its importance in natural infection, RTs are also widely used in biotechnology for their capacity to synthesize complementary DNA using RNA as templates. Intrinsic fidelity of RT’s DNA-dependent DNA synthesis has been extensively studied. In M13mp2 lacZα forward mutation assays, WT HIV-1 RTs of group M/subtype B showed >10-fold higher error rates than murine leukemia virus (MLV) and avian myeloblastosis virus (AMV) RTs. This Thesis aimed to determine error rates for RNA-dependent DNA synthesis catalyzed by several RTs, including WT HIV-1BH10, HIV-1ESP49, AMV and MLV RTs, and the high-fidelity mutants of HIV-1ESP49 RT K65R and K65R/V75I. Our results show that these retroviral RTs have less than 2-fold differences in fidelity, with error rates in the range of 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, needed to synthesize the RNA template. Despite assay limitations, we concluded 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. Data provided by deep sequencing experiments also showed similar differences in accuracy between HIV-1BH10 and K65R/V75IESP49 RTs.