Do nucleoside analog -resistance mutations increase the fidelity of human immunodeficiency virus type-1 (HIV -1) reverse transcriptase?

Abstract

The human immunodeficiency virus type 1 (HIV-1) viral enzyme reverse transcriptase (RT) is responsible for replicating the HIV genome. HIV-RT mediates the conversion of the viral RNA genome into double stranded DNA. We found that the K65R mutation decreased the mutation rate by 8-fold, but the L74V mutation, which directly contacts the templating base, had a mutation frequency very similar to wild type. A large number of the mutant plaques identified from the assay were sequenced. Studying the distribution of errors that the L74V RT made on a lacZalpha template indicated that this mutation in RT results in an enzyme that is more prone to make frameshift errors than the wild type RT.;Long-term treatment with anti-retroviral treatments leads to novel mutations within the fingers subdomain that confers resistance to multi-dideoxynucleoside analogs (MDR). Previously, characterized multi-drug resistant viruses included mutations such as M184V, K65R, and a specific set of mutations that were distributed in the finger and palm subdomains (Q151M, A62V, V75I, F77L, and F116Y). The novel mutations associated with MDR included insertions of up to 15 amino acids between residues 68--70 and an amino acid substitution at residue 69 (T69S). The majority of insertions are the dipeptide insertions of the type AG, SG, and SS. These insertions are commonly found in a background of classic AZT resistant mutations, such as M41L and T215Y, or a second class of MDR mutations A62V and Q151M. We selected genotypes that occur most commonly including T69S-AG (AG, SG, and SS), T69S-SG, and T69S-SS alone or in a combination with a set of AZT resistant mutations M41L, L210W, R21LLK, L214F, T215Y (MAG AZ and MSGAZ) or an alternate set of mutations where the M41L is replaced by the MDR mutation A62V (VAGAZ, VSGAZ, and VSSAZ). We showed that each of the sets of mutations displayed a decreased efficiency of misincorporation. This result was further corroborated by a decrease in the overall mutation frequency of the mutant RT (4--16 fold), with three of the mutants displaying the largest decreases in mutation frequency ever reported for naturally occurring RT mutations (11-fold, 15-fold, and 16-fold respectively for (VAGAZ, MAGAZ, and MSG AZ). (Abstract shortened by UMI.).