Mechanistic studies to understand the progressive development of resistance in human immunodeficiency virus type 1 reverse transcriptase to abacavir.

Abstract:

Abacavir has been shown to select for multiple resistant mutations in the human immunodeficiency type 1 (HIV-1) pol gene. In an attempt to understand the molecular mechanism of resistance in response to abacavir, and nucleoside analogs in general, a set of reverse transcriptase mutants were studied to evaluate their kinetics of nucleotide incorporation and removal. It was found that, similar to the multidrug-resistant mutant reverse transcriptase (RT)(Q151M), the mutations L74V, M184V, and a triple mutant containing L74V/Y115F/M184V all caused increased selectivity for dGTP over the active metabolite of abacavir (carbovir triphosphate). However, the magnitude of resistance observed in cell culture to abacavir in previous studies was less than that observed to other compounds. Our mechanistic studies suggest that this may be due to carbovir triphosphate decreasing the overall effect on its efficiency of incorporation by forming strong hydrophobic interactions in the RT active site. Unlike RT(AZTR), no increase in the rate of ATP- or PP(i)-mediated chain terminator removal relative to RT(WT) could be detected for any of the mutants. However, marked decreases in the steady-state rate may serve as a mechanism for increased removal of a chain-terminating carbovir monophosphate by increasing the time spent at the primer terminus for some of the mutants studied. The triple mutant showed no advantage in selectivity over RT(M184V) and was severely impaired in its ability to remove a chain terminator, giving no kinetic basis for its increased resistance in a cellular system. Biochemical properties including percentage of active sites, fidelity, and processivity may suggest that the triple mutant's increased resistance to abacavir in cell culture is perhaps due to a fitness advantage, although further cellular studies are needed to verify this hypothesis. These data serve to further the understanding of how mutations in RT confer resistance to nucleoside analogs.

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