Abstract
BackgroundWe investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT).MethodsRecombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments.ResultsThe efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT.ConclusionThe diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.
Highlights
We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT)
Our experiments have revealed that subtype C human immunodeficiency virus type 1 (HIV-1) RT has similar enzymatic activity to subtype B RT, and that the K65R and K65R+M184V mutations, affect subtype C RT function in a manner similar to that seen with subtype B RT
Specific effects include: 1) The efficiency of ssDNA synthesis and initiation is reduced; 2) At low dNTP concentration, K65R RT exhibited lower activity in singlecycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. 3) the discrimination of nucleotides is equivalently reduced in subtype C RT as in subtype B RT; and 4) the excision of incorporated nucleotides is decreased in a similar fashion in both RTs, in agreement with previous results [9,19,21,22,23]
Summary
We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT). Genetic divergence in the RT enzyme may be linked to differential acquisition of resistance to nucleoside or nucleotide RT inhibitors (N(t)RTIs) that are core constituents of antiretroviral (ARV) regimens for treatment of HIV-1 infection. These drugs include the eight N(t)RTIs approved for clinical treatment of HIV-1 infection: zidovudine (ZDV), stavudine (d4T), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), abacavir (ABC), emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) [6]
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