Xu Hong-Tao, Martinez-Cajas Jorge L, Ntemgwa Michel L, Coutsinos Dimitrios, Frankel Fernando A, Brenner Bluma G, Wainberg Mark A
McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec H3T1E2, Canada.
Retrovirology. 2009 Feb 11;6:14. doi: 10.1186/1742-4690-6-14.
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).
Recombinant 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.
The 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.
The 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.
我们研究了K65R突变以及K65R加M184V突变对C亚型逆转录酶（RT）酶功能及耐药机制的影响。
从大肠杆菌中纯化出含有K65R或K65R + M184V的重组C亚型HIV-1 RT。在无细胞试验中测定野生型（WT）和两种亚型突变RT的酶活性及替诺福韦（TFV）掺入效率。使用以HIV-1 PBS RNA为模板、tRNA3Lys为引物的基于凝胶的试验，测量C亚型RT的（-）单链DNA合成及起始效率。在不同的脱氧核苷三磷酸（dNTP）浓度下测定单轮持续合成能力。进行稳态分析以测量二磷酸替诺福韦（TFV-DP）的相对抑制能力（ki/km）。在时间进程实验中监测ATP依赖的TFV或齐多夫定（ZDV）终止的DNA合成的切除及挽救情况。
C亚型RT以tRNA为引物的（-）单链DNA合成效率为：WT > K65R > K65R + M184V RT。在低dNTP浓度下，K65R RT在单轮持续合成能力试验中表现出较低活性，而K65R + M184V突变体显示出与dNTP浓度无关的持续合成能力降低。与WT RT相比，K65R和K65R + M184V RT的ATP介导的TFV或ZDV终止引物的切除减少。与WT RT相比，K65R和K65R + M184V对TFV-DP的半数抑制浓度（IC50）分别增加了9.8倍和5倍。与WT RT相比，K65R和K65R + M184V的TFV的Ki/Km分别增加了4.1倍和7.2倍。
对于C亚型以及B亚型，已证实含K65R的RT在低dNTP浓度下起始效率降低。尽管切除减少，但这种结合/掺入的降低导致K65R和K65R + M184 RT对TFV-DP敏感性降低。
