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Analysis of selection pressure and mutational pattern of HIV type 1 reverse transcriptase region among treated and nontreated patients.

ABSTRACT: Variation of the HIV-1 subtype C reverse transcriptase region (RT) resulting in response to the selective pressures of drug therapy remains poorly characterized. Here, we compared the genetic variation resulting in the presence and absence of antiretroviral drug selective pressures on HIV-1 subtype C RT among nontreated and treated patients. The nucleotide variability, nonsynonymous and synonymous ratio, and the positively selected mutations were determined by comparing the RT sequences isolated at two time points among nontreated (baseline and follow-up) and treated patients (baseline and treatment failure). Compared to the nontreated patients, the intrapatient nucleotide variability, the number of nonsynonymous and synonymous substitutions was significantly higher among the treated patients. Among the mutations positively selected, the frequency of D121Y, I135R, and Q207E increased and the frequency of mutation S48T decreased significantly during treatment failure. Further studies are essential to discover the role of these mutations during treatment in HIV-1 subtype C.

SUBMITTER: Shanmugasundaram U

PROVIDER: S-EPMC5206687 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Analysis of selection pressure and mutational pattern of HIV type 1 reverse transcriptase region among treated and nontreated patients.

Shanmugasundaram Uma U Solomon Suniti S Madhavan Vidya V Kailapuri Murugavel G MG Nagalingeswaran Kumarasamy K Solomon Sunil Suhas SS Mayer Kenneth H KH Pachamuthu Balakrishnan B

AIDS research and human retroviruses 20100919 10

Variation of the HIV-1 subtype C reverse transcriptase region (RT) resulting in response to the selective pressures of drug therapy remains poorly characterized. Here, we compared the genetic variation resulting in the presence and absence of antiretroviral drug selective pressures on HIV-1 subtype C RT among nontreated and treated patients. The nucleotide variability, nonsynonymous and synonymous ratio, and the positively selected mutations were determined by comparing the RT sequences isolated ...[more]

PMID: 20849301

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Project description:Amino acid Lys(65) is part of the highly flexible beta3-beta4 loop in the fingers domain of the 66 kDa subunit of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Recent crystal data show that the epsilon-amino group of Lys(65) interacts with the gamma-phosphate of the bound deoxynucleoside triphosphate ('dNTP') substrate [Huang, Chopra, Verdine and Harrison (1998) Science 282, 1669-1675]. In order to biochemically define the function of RT Lys(65), we have used site-specific mutagenesis to generate RT with a variety of substitutions at this position, including K65E, K65Q, K65A and K65R. Kinetic analyses demonstrate that if Lys(65) in RT is substituted with an amino acid other than arginine the enzyme exhibits dramatic decreases in the binding affinity (K(m)) for all dNTP substrates, in RT catalytic efficiency (k(cat)/K(m)) and in the mutant enzyme's ability to carry out pyrophosphorolysis, the reverse reaction of DNA synthesis. The pH optimum for the DNA polymerase activity of K65E RT was 6.5, compared to 7.5 for the wild-type enzyme, and 8.0 for the K65R, K65A and K65Q mutants. Molecular modelling studies show that mutations of Lys(65) do not affect the geometry of the loop's alpha-carbon backbone, but rather lead to changes in positioning of the side chains of residues Lys(70) and Arg(72). In particular, Glu in K65E can form a salt bridge with Arg(72), leading to the diminution of the latter residue's interaction with the alpha-phosphate of the dNTP residue. This alteration in dNTP-binding may explain the large pH-dependent changes in both dNTP-binding and catalytic efficiency noted with the enzyme. Furthermore, the K65A, K65Q and K65E mutant enzymes are 100-fold less sensitive to all dideoxynucleoside triphosphate ('ddNTP') inhibitors, whereas the K65R mutation results in a selective 10-fold decrease in binding of ddCTP and ddATP only. This implies that mutations at position 65 in HIV-1 RT influence the nucleotide-binding specificity of the enzyme.

Project description:N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. Biochemical studies demonstrated that N348I indirectly increases AZT resistance by decreasing the frequency of secondary ribonuclease H (RNase H) cleavages that reduce the RNA/DNA duplex length of the template/primer (T/P) and diminish the efficiency of AZT-monophosphate (MP) excision. By contrast, there is some discrepancy in the literature in regard to the mechanisms associated with nevirapine resistance: one study suggested that it is due to decreased inhibitor binding while others suggest that it may be related to the decreased RNase H cleavage phenotype. From a structural perspective, N348 in both subunits of RT resides distal to the enzyme's active sites, to the T/P binding tract and to the nevirapine-binding pocket. As such, the structural mechanisms associated with the resistance phenotypes are not known.Using a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the ?14-?15 loop in the p51 subunit of RT and the RNA template. Substitution of the asparagine at codon 348 in the p51 subunit with either isoleucine or leucine abrogated the observed protein-RNA interaction, thus, providing a possible explanation for the decreased RNase H phenotype. By contrast, alanine or glutamine substitutions exerted no effect. To validate this model, we introduced the N348I, N348L, N348A and N348Q mutations into RT and purified enzymes that contained subunit-specific mutations. N348I and N348L significantly decreased the frequency of secondary RNase H cleavages and increased the enzyme's ability to excise AZT-MP. As predicted by the modelling, this phenotype was due to the mutation in the p51 subunit of RT. By contrast, the N348A and N348Q RTs exhibited RNase H cleavage profiles and AZT-MP excision activities similar to the wild-type enzyme. All N348 mutant RTs exhibited decreased nevirapine susceptibility, although the N348I and N348L mutations conferred higher fold resistance values compared to N348A and N348Q. Nevirapine resistance was also largely due to the mutation present in the p51 subunit of RT.This study demonstrates that N348I-mediated AZT and nevirapine resistance is due to the mutation in the p51 subunit of RT.

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Analysis of selection pressure and mutational pattern of HIV type 1 reverse transcriptase region among treated and nontreated patients.

Publications

Analysis of selection pressure and mutational pattern of HIV type 1 reverse transcriptase region among treated and nontreated patients.

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