AEGiS-11IAC: Mutations associated with 3TC-resistance enhance the polymerase fidelity of HIV-1 RT.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Mutations associated with 3TC-resistance enhance the polymerase fidelity of HIV-1 RT.

Int Conf AIDS 1996 Jul 7-12; 11:11 (abstract no. Mo.A.381)
Prasad VR, Drosopoulos W; Dept. of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY, USA. Fax: 718-430-8711. E-mail: Prasad@aecom.yu.edu Vinayaka.

OBJECTIVE: To determine whether mutations associated with 3TC-resistance confer an increased polymerase fidelity to HIV-1 reverse transcriptase.

METHODS: Recombinant wild type, Met184Val, Glu89Gly and Met184Val/Glu89Gly HIV-1 RT heterodimers were purified. These RTs were assayed in vitro, with model template-primers, for their ability to misinsert dNTPs as well as to extend primers with mismatched termini. The reaction products were separated on polyacrylamide gels, intensities of extended and unextended bands determined, and used to calculate the Km for dNTP and the maximal velocity of the reaction (Vmax). The Km and Vmax were used to determine the efficiencies of misinsertion (fins) and mispair extension (fext).

RESULTS: With respect to misinsertion, our results show that both Glu89Gly and Met184Val variant RTs display an increase in dNTP insertion fidelity. Depending upon the type of mispair studied, a 2 to 17-fold increase over wild type levels was observed in insertion fidelity for both the enzymes. However, the type of misinsertions for which an increase in fidelity was observed were not identical between the two enzymes. The overall increase in misinsertion fidelity over the wild type enzyme was 3.1-fold for the Met184V al RT and 5.9-fold for Glu89Gly variant RTs respectively. Preliminary results show that Met 184Val, Glu89Gly and the double mutant Met184Val/Glu89Gly display an increase in mispair extension fidelity.

CONCLUSIONS: Our results show that two mutations, both shown to be associated with 3TC-resistance, independently increase the polymerase fidelity of HIV-1 RT. The decrease in the rate of errors via two major pathways, suggests that the overall increase in fidelity must be considerably higher for these mutants. Thus, an ability select for a high-fidelity virus could be used as a tool to reduce or the slowing the appearance of resistance to a secondary drug in combination therapies as well as to decrease the rate of emergence of immune-escape mutants.

Keywords: AEGIS, Lamivudine, HIV-1 Reverse Transcriptase, HIV-1, Mutation, Reverse Transcriptase Inhibitors, Templates, Genetic, Anti-HIV Agents, DNA Primers, Variation (Genetics), In Vitro, genetics, ICA11

960707
MoA381