Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance.
BACKGROUND
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Xu
Hong-Tao
HT
McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec H3T1E2, Canada. hongtaoxu_00@yahoo.com
Martinez-Cajas
Jorge L
JL
Ntemgwa
Michel L
ML
Coutsinos
Dimitrios
D
Frankel
Fernando A
FA
Brenner
Bluma G
BG
Wainberg
Mark A
MA
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D024882:Drug Resistance, Viral; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D020125:Mutation, Missense; D063065:Organophosphonates / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D048348:Reverse Transcription; D000068698:Tenofovir
D017931:DNA Primers; D063065:Organophosphonates; D012367:RNA, Viral; D000068698:Tenofovir; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D000225:Adenine