ATP-dependent removal of nucleoside reverse transcriptase inhibitors by human immunodeficiency virus type 1 reverse transcriptase

Abstract

Removal of nucleoside chain terminator inhibitors mediated by human immunodeficiency virus (HIV) reverse transcriptase (RT) using ATP as an acceptor molecule has been proposed as a novel mechanism of HIV resistance. Recombinant wild-type and mutant HIV type 1 (HIV-1) RT enzymes with thymidine analog resistance mutations D67N, K70R, and T215Y were analyzed for their ability to remove eight nucleoside reverse transcriptase inhibitors in the presence of physiological concentrations of ATP. The order for the rate of removal of the eight inhibitors by the mutant RT enzyme was zidovudine (AZT) > stavudine (d4T) >> zalcitabine (ddC) > abacavir > amdoxovir (DAPD) > lamivudine (3TC) > didanosine (ddI) > tenofovir. Thymidine analogs AZT and d4T were the most significantly removed by the mutant enzyme, suggesting that removal of these inhibitors by the ATP-dependent removal mechanism contributes to the AZT and d4T resistance observed in patients with HIV expressing thymidine analog resistance mutations. ATP-dependent removal of tenofovir was 22- to 35-fold less efficient than removal of d4T and AZT, respectively. The addition of ATP and the next complementary deoxynucleoside triphosphate caused a reduction of ATP-mediated removal of d4T, ddC, and DAPD, while AZT and abacavir removal was unaffected. The reduction of d4T, ddC, and DAPD removal in the presence of the deoxynucleoside triphosphate could explain the minor changes in susceptibility to these drugs observed in conventional in vitro phenotypic assays using cells that have higher deoxynucleoside triphosphate pools. The minimal removal of abacavir, ddC, DAPD, 3TC, ddI, and tenofovir is consistent with the minor changes in susceptibility to these drugs observed for HIV mutants with thymidine analog resistance mutations.

FIG. 1.

ATP-dependent removal of chain terminator inhibitors by mutant RT. Removal of AZT, d4T, ddC, 3TC, carbovir, DXG, ddA, and tenofovir at 0, 10, and 20 min following addition of 3.2 mM ATP both in the absence and presence (+dNTP) of a 50 μM concentration of the next complementary dNTP as indicated.

FIG. 2.

Comparison of NRTI removal by mutant RT. Shown is removal of NRTIs AZT (open circle), d4T (filled inverted triangle), ddC (filled square), carbovir (open diamond), DXG (filled diamond), 3TC (open square), ddA (open triangle), and tenofovir (filled circle) averaged from two or three experiments with standard deviations following addition of 3.2 mM ATP in the absence of the next complementary dNTP.

FIG. 3.

Effect of the next complementary dNTP on ATP-dependent removal. Shown is removal of AZT, d4T, ddC, 3TC, carbovir, DXG, ddA, and tenofovir averaged from two or three experiments with standard deviations in the presence (open circles) and absence (filled circles) of 50 μM concentrations of the next complementary dNTP. Note that the y axes for the AZT and d4T graphs extend from 0 to 30% while those for the ddC, 3TC, carbovir, DXG, ddA, and tenofovir graphs extend from 0 to 8%.

FIG. 4.

Analysis of AZT, d4T, and tenofovir ATP-dependent removal by wild-type and mutant RT. Shown is removal of tenofovir, AZT, and d4T from tenofovir-, AZT-, and d4T-terminated primers/templates by wild-type RT and the RT mutant with D67N, K70R, and T215Y RT mutations following the addition of 3.2 mM ATP.