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, 51 (1), 48-53

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

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Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

Nicolas Sluis-Cremer et al. Antimicrob Agents Chemother.

Abstract

The K70E mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has become more prevalent in clinical samples, particularly in isolates derived from patients for whom triple-nucleoside regimens that include tenofovir (TNV), abacavir, and lamivudine (3TC) failed. To elucidate the molecular mechanism by which this mutation confers resistance to these nucleoside RT inhibitors (NRTI), we conducted detailed biochemical analyses comparing wild-type (WT), K70E, and K65R HIV-1 RT. Pre-steady-state kinetic experiments demonstrate that the K70E mutation in HIV-1 RT allows the enzyme to discriminate between the natural deoxynucleoside triphosphate substrate and the NRTI triphosphate (NRTI-TP). Compared to the WT enzyme, K70E RT showed 2.1-, 2.3-, and 3.5-fold-higher levels of resistance toward TNV-diphosphate, carbovir-TP, and 3TC-TP, respectively. By comparison, K65R RT demonstrated 12.4-, 12.0-, and 13.1-fold-higher levels of resistance, respectively, toward the same analogs. NRTI-TP discrimination by the K70E (and K65R) mutation was primarily due to decreased rates of NRTI-TP incorporation and not to changes in analog binding affinity. The K65R and K70E mutations also profoundly impaired the ability of RT to excise 3'-azido-2',3'-dideoxythymidine monophosphate (AZT-MP) and other NRTI-MP from the 3' end of a chain-terminated primer. When introduced into an enzyme with the thymidine analog mutations (TAMs) M41L, L210W, and T215Y, the K70E mutation inhibited ATP-mediated excision of AZT-MP. Taken together, these findings indicate that the K70E mutation, like the K65R mutation, reduces susceptibility to NRTI by selectively decreasing NRTI-TP incorporation and is antagonistic to TAM-mediated nucleotide excision.

Figures

FIG. 1.
FIG. 1.
Isotherms for ATP (left-hand panel)- or PPi (right-hand panel)-mediated phosphorolytic excision of AZT-MP (A), 3TC-MP (B), CBV-MP (C), and TNV (D) carried out by WT (ο), K65R (□), or K70E (▿) HIV-1 RT. None of the enzymes exhibited ATP-mediated excision of 3TC-MP (data not shown). Data are the means of three or four independent experiments.
FIG. 2.
FIG. 2.
ATP-mediated excision of AZT-MP and rescue of DNA synthesis carried out by WT, Aztr (M41L/L210W/T215Y), and Aztr K70E RT. (A) ATP-mediated unblocking reaction analyzed by denaturing gel electrophoresis. Excision products were analyzed at 4, 8, 12, 16, 20, 40, 60, and 90 min. (B) Isotherms for ATP-mediated phosphorolytic excision of AZT-MP by WT (ο), Aztr (▿), and Aztr K70E (□) HIV-1 RT. Data are the means of three independent experiments.

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