Molecular basis of antagonism between K70E and K65R tenofovir-associated mutations in HIV-1 reverse transcriptase

The K70E mutation in HIV-1 reverse transcriptase was observed in 10% of virologic non-responders of the abacavir/lamivudine/tenofovir arm of ESS30009, alone, or in mixtures with K65R by population sequencing. Clonal analysis of six ESS30009 K70E isolates failed to identify double mutants carrying K65R + K70E. Site-directed K70E mutants had a replication capacity of 97 ± 29%, but only 2.4 ± 0.9% for K65R + K70E and 0.01% for K65R + K70E + M184V mutants. K65R + K70E phenotypic fold changes for abacavir, lamivudine and tenofovir were comparable to reported values for K65R alone. In molecular dynamic simulations, the ɛ-amino group of K65 was positioned 2.7 ± 0.1 Å from the γ-phosphate of the dTTP ligand and stabilized the triphosphate. In the R65 mutant, this distance increased to 4.2 ± 0.4 Å and the interaction energy with the ligand was less favorable, but the K70 ɛ-amino group was repositioned closer to the γ-phosphate and had a more favorable interaction energy. In the double mutant, E70 could not stabilize the γ-phosphate, resulting in a more severe defect. The net effect of the atomic-level changes in the double mutant may be to destabilize the pyrophosphate leaving group of the ligand, more severely affecting the catalytic rate of the polymerization reaction than the R65 single mutation.