Diseño, purificación y caracterización de variantes de la retrotranscriptasa del virus de la inmunodeficiencia humana de interés biotecnológico

Abstract

Retroviruses are RNA viruses that replicate through a DNA intermediate, in a process catalyzed by the viral reverse transcriptase (RT). Retroviral RTs are multifunctional enzymes with RNA- and DNA-dependent DNA polymerase, endonuclease (RNase H), strand transfer and strand displacement activities, which catalyze the synthesis of proviral DNA using the viral RNA as template. RTs are extensively used in recombinant DNA technology to synthesize cDNA from messenger RNA. However, the presence of secondary structures in RNA templates can limit the efficiency of reverse transcription. This problem can be mitigated by using RTs active at high temperatures. Murine leukemia virus (MLV) RT and avian myeloblastosis virus (AMV) RT are frequently used in biotechnological applications, but their thermal stability is limited. Human immunodeficiency virus type 1 (HIV-1) RTs have higher stability at elevated temperatures, but they are less faithful than the MLV RT. In this Doctoral Thesis we aimed to study fidelity differences between RTs of MLV, xenotropic murine leukemia virus-related virus (XMRV) and HIV-1 variants, and improve the fidelity of HIV-1 group O RTs by protein engineering. The fidelity of DNA synthesis of XMRV RT was found to be similar to that shown by the MLV RT. Both oncorretroviral RTs were >13 times more faithful than an HIV-1 group M subtype B RT variant designated as BH10_WT (wild-type BH10). In M13mp2-based forward mutation assays, the wild-type HIV-1 group O RT (designated as O_WT RT) showed 2.5-fold increased accuracy in comparison with the BH10_WT RT. Nevertheless, MLV and XMRV RTs were about 5 to 6 times more faithful than the O_WT RT. In this Thesis, we have examined the effects of mutations K65R and R78A on the thermal stability and fidelity of DNA synthesis of HIV-1 group O RT in the presence or absence of V75I. Our results showed that K65R and K65R/V75I do not affect the thermal stability of the enzyme, but increase its accuracy to similar levels as the MLV RT. These HIV-1 group O RT mutants were >9 times more faithful than the O_WT enzyme as determined in forward mutation assays a using DNA template. Misinsertion and mispair extension kinetic studies revealed that the selection of nucleotides is governed by kpol in the case of O_K65R RT and kpol and Kd in the case of O_K65R/V75I RT. We have used an M13mp2-based assay to determine the fidelity of RNA-dependent synthesis of DNA. The error rates of wild-type and mutant enzymes were determined. These assays demonstrated that mutations K65R and K65R/V75I increased the fidelity of RNA-dependent DNA polymerization. However, the effects of the mutations were less pronounced than with DNA templates due to the intrinsic error rate of the T7 RNA polymerase used for synthesis of the RNA template.