Molecular mechanisms of bidirectional antagonism between K65R and thymidine analog mutations in HIV-1 reverse transcriptase

Abstract

The K65R mutation in HIV-1 reverse transcriptase (RT) decreases susceptibility to all approved nucleoside reverse transcriptase inhibitors (NRTI) except zidovudine by selectively decreasing the incorporation of the NRTI triphosphate compared with the natural deoxyribonucleotide triphosphate substrate. Thymidine analog mutations (TAMs) confer high-level resistance to zidovudine and cross-resistance to other NRTI by increasing excision of the chain-terminating NRTI monophosphate via a phosphorolytic cleavage reaction. Recent virology and genetic studies have shown bidirectional antagonism between K65R and TAMs. The aim of this study was to elucidate the biochemical and structural mechanisms responsible for this antagonism. Steady-state and pre-steady-state kinetic analyses of NRTI triphosphate incorporation and NRTI monophosphate excision by RT containing K65R or TAMs were conducted and complemented by molecular modeling. The addition of K65R to two clinically relevant combinations of TAMs (M41L/L210W/T215Y or D67N/K70R/T215F/K219Q) significantly reduced the recombinant enzymes' ability to excise all chain-terminating NRTI monophosphate. Transient kinetic analyses showed that TAMs decreased the extent to which RT containing K65R could discriminate against D-nucleotide analogs, but not L-nucleotide analogs, by partly restoring the maximum rate of NRTI triphosphate incorporation. In addition, the TAMs combination D67N/K70R/T215F/K219Q decreased susceptibility to the L-nucleotide lamivudine by a discrimination mechanism, whereas the M41L/L210W/T215Y combination had little effect on susceptibility to lamivudine. K65R antagonizes the NRTI monophosphate excision activity of RT containing TAMs. TAMs antagonize the ability of K65R RT to discriminate against the nucleotide analog. Therapies including NRTI that select for both TAMs and K65R may prolong treatment response through the mutually antagonistic interactions between these resistance mutations.