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 imediated 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.

Mutations M184V and Y115F in HIV-1 Reverse Transcriptase Discriminate against “Nucleotide-competing Reverse Transcriptase Inhibitors”

“…The differences in K d (app) values are in good agreement with enzyme kinetic measurements that point to decreases in K d and K m values with the Y115F mutant (21,22). The Y115F mutation is seen, infrequently, in conjunction with M184V in isolates of patients treated with abacavir (9,23).…”

“…The Y115F mutation is seen, infrequently, in conjunction with M184V in isolates of patients treated with abacavir (9,23). However, Y115F does not appear to further diminish the efficiency of incorporation of the NRTI (21).…”

Computational model of hepatitis B virus DNA polymerase: Molecular dynamics and docking to understand resistant mutations

“…7,8 Although there is considerable similarity between the two aromatic amino acids, Tyr and Phe, the substitution could still be significant in this case, since studies have shown that this substitution in combination with L74V mutation confers HIV-1 RT resistance against abacavir. 21 Our final model included loops of length 26, 11, and 9 residues, whereas the model of Das et al 7 had corresponding loops of 26 and 23 residues, while Bartholomeuz et al 8 had one long loop of 46 residues. Figure 3.…”

Thymidine Analogue Resistance Suppression by V75I of HIV-1 Reverse Transcriptase

“…10 and 11). Thus, viral RTs bearing mutations A62V, V75I, F77L, F116Y, and Q151M showed decreased discrimination efficiencies against the triphosphorylated forms of thymidine analogues, such as zidovudine (␤-D-(ϩ)-3Ј-azido-3Ј-deoxythymidine (AZT)) or stavudine (␤-D-(ϩ)-2Ј,3Ј-didehydro-2Ј,3Ј-dideoxythymidine (d4T)), as well as didanosine, zalcitabine, and abacavir (6,12,13). These observations were in agreement with the high level resistance to those inhibitors shown in phenotypic assays by HIV variants carrying those mutations in their RTs (14,15).…”