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12th International HIV Drug Resistance Workshop10–14 June 2003, Cabo del Sol, Los Cabos, Mexico |
BACKGROUND: The insertion of two amino acids after residue 69 of HIV-1 reverse transcriptase (RT) is a rare mutation that may develop in viruses containing multiple thymidine analogue-associated mutations (TAMs) and confers high-level resistance to all currently approved chain-terminating nucleoside and nucleotide RT inhibitors (NRTIs). Decreased incorporation and increased excision are two known mechanisms of NRTI resistance. Excision for many NRTIs may be inhibited by binding of the nucleotide complementary to the next position in the template (next nt) to form a stable dead-end complex where the chainterminator is protected from excision. The mechanism of resistance to tenofovir for RT with an insertion mutation and multiple TAMs was examined in this study.
METHODS: A patient-derived HIV-1 virus (FS-SSS) was obtained that contained the SS insertion after residue 69 in a background of additional resistance mutations M41L, T69S, L74V, L210W and T215Y. The insertion and T69S were reverted by site-directed mutagenesis in a second virus (FS) that retained the other resistance mutations. In vitro drug susceptibility was determined by the PhenoSense assay. Incorporation of tenofovir was examined by measuring steady-state kinetic constants for wild-type and mutant RTs. ATP-mediated excision with or without the next nt was measured. Molecular models and molecular dynamics simulations were produced using Sybyl software.
RESULTS: The multiple TAM-containing FS virus exhibited a large reduction in zidovudine susceptibility and smaller reductions in susceptibility to other NRTIs including tenofovir; the FS-SSS virus showed greater reductions in susceptibility to all NRTIs including tenofovir. The relative binding/incorporation of tenofovir diphosphate was slightly decreased for FS-SSS RT (2.8-fold) compared to wild-type, but not significantly for FS RT (1.7-fold). However, significant ATP-mediated excision of tenofovir was detected for both mutant RTs with FS-SSS > FS > wild-type, and excision rates of 3.5, 1.8 and 0.8% per min, respectively. The presence of physiological concentrations of the next nt inhibited tenofovir excision by wild-type, slightly inhibited excision by FS, whereas excision by FS-SSS remained high (12-fold greater than wild-type). Computer modelling shows that the insertion mutation could generate a more flexible β3-β4 fingers loop domain that would enhance excision by facilitating dissociation of the ATP-mediated excision product and/or destabilizing the protective next nt complex.
CONCLUSIONS: Increased ATP-mediated excision of incorporated tenofovir without efficient inhibition by the next nucleotide appears to be the primary mechanism of tenofovir resistance for HIV-1 RT with T69 insertion mutations and multiple TAMs. Decreased binding/incorporation of tenofovir also makes a minor contribution to tenofovir resistance. The same TAMs without the insertion mutation showed detectable, but lower levels of excision and greater inhibition by the next nucleotide. Increased flexibility of the β3-β4 loop by the insertion mutation may be the basis for the high-level and broad NRTI cross resistance caused by the T69 insertion mutations.
PRESENTING AUTHOR: MD Miller
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2003-07-08
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