Determinantes moleculares implicados en la resistencia a análogos a timidina y en la fidelidad de copia de la retrotranscriptasa del virus de la inmunodeficiencia humana tipo 1

Abstract

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a multifunctional enzyme with RNA- and DNA-dependent DNA polymerase, RNase H, strand transfer and strand displacement activities. HIV-1 RT is a heterodimer enzyme composed of two subunits, p66 and p51. Reverse transcription is error prone and contributes to the high genetic variability of retroviruses. One of the consequences of the high mutation rates has been the emergence of drug-resistant HIV variants. With the increasing complexity of the antiretroviral regimens, novel mutational patterns conferring high-level resistance to nucleoside RT inhibitors have been identified in viral isolates, among them, insertions in the β3-β4 hairpin-loop of HIV-1 RT. The combination of a dipeptide insertion and thymidine analogues mutations in the RT confers an ATP-dependent phosphorolytic activity that facilitates the removal of the inhibitor from primers terminated with AZT or d4T. One of the objectives of this Thesis has been the identification of molecular determinants involved in the excision activity of HIV-1 RT containing a dipeptide insertion in the β3-β4 hairpin loop. The β3-β4 hairpin loop plays an important role in nucleoside analogue resistance, and modulates the RT fidelity because of its interactions with the template-primer. We have studied the role of Val-75 in fidelity of DNA synthesis under steady-state and pre-steadystate conditions and using M13 lacZα genetic assays. In this Doctoral Thesis we report that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and d4T resensitization through the loss of the ATP-mediated removal activity. The results suggest that Tyr-215 is involved in the effective positioning of the PPi donor for the catalytic attack of the terminated primer. We also show that oxygen atoms bound to de γ phosphate group of nucleoside triphosphates are important for the efficiency of the excision reaction. Furthermore, the blocked primer after incorporation of an α-phosphorothioate derivative of AZT becomes resistant to excision by insertion-containing RTs, which contain high levels of excision activity. These results suggest that phosphorothioate derivatives of currently approved drugs could be useful against excision-proficient HIV-1 RTs. Finally, we report that the fidelity of RTs bearing mutations V75F and V75I increased 1.8- and 3-fold, respectively, as measured by the M13 lacZα forward mutation assay, while V75A showed 1.4-fold decreased accuracy. Steady- and pre-steady state kinetics demonstrated that the increased fidelity of V75I and V75F was related to their decreased ability to extend mismatched template–primers, while misincorporation efficiencies were not significantly affected by mutations. Altogether, these data suggest that Val-75 is an important determinant of fidelity in HIV-1 RT.