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Background:  HIV-1 reverse transcriptase (RT) thymidine analog mutations (TAM), selected by AZT and d4T, can cause reduced susceptibility to all approved NRTI. TAM can occur in 2 distinct genotypic patterns (D67N+K70R+T215F+K219Q/E/N or M41L+D67N+L210W+T215Y), where only the latter pattern causes reduced susceptibility to tenofovir in vitro and in patients. This study characterizes the molecular mechanisms by which M41L+D67N+L210W+T215Y RT mutant HIV-1 causes reduced susceptibility to tenofovir and other NRTIs.

Methods:  Drug susceptibilities in vitro were determined for wild type and the M41L+D67N+L210W+T215Y (designated 4Y) site-directed mutant HIV-1. To study the mechanisms of resistance to the adenosine analogs tenofovir, ddI, and AZA (the adenosine analog of AZT), as well as AZT, the steady state kinetic constants K_i and K_m and efficiency of ATP-mediated excision after incorporation were determined for wild type and 4Y RT enzymes. The translocational equilibrium of incorporated tenofovir, ddI, AZA, and AZT were measured by site-specific footprinting for both enzymes.

Results:  The 4Y virus had 3-fold reduced susceptibility to tenofovir, was highly resistant to AZT (>126-fold), but appeared susceptible to ddI (1-fold) compared to wild type. The K_i/K_m values for the triphosphates of AZT, tenofovir, ddI (ddA), and AZA were similar to wild type. ATP-mediated excision of AZT, AZA, tenofovir, and ddA followed the order AZT > AZA > tenofovir  ddA for wild type RT. The excision of AZT, AZA, and tenofovir by the 4Y RT were significantly greater than wild type RT, while incorporated ddA remained almost uncleaved. The excision of NRTI can only take place when the incorporated NRTI resides in the pre-translocation site. For both wild type and 4Y RT, in the absence of the next nucleotide, the pre-translocation complex was favored for AZT-, AZA-, and tenofovir-terminated primers. For primers terminated with ddA there was no preference for the pre- or post-translocation complex.

Conclusions:  The decreased susceptibilities to NRTI by this 4-TAM RT correlate with the rates of excision of NRTI: AZT > AZA > tenofovir  ddA. In the absence of a change in binding/incorporation for these drugs, the increased excision rate by this mutant RT is implicated as the mechanism of resistance to AZA, AZT, and tenofovir. This conclusion is supported by the translocational equilibrium footprints that suggest that incorporated AZA, AZT, and tenofovir are preferentially located in the pre-translocation site.

Keywords: reverse transcriptase; tenofovir; TAMs