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13th International HIV Drug Resistance Workshop8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain |
BACKGROUND: HIV-1 reverse transcriptase (RT) is an error-prone enzyme, which frequently incorporates mismatched nucleotides. In view of the fact that G:T mispairs are the easiest to form, we asked whether thymidine analogues may likewise be incorporated opposite template G. Such a scenario may affect susceptibility to these drugs in the context of both wild -type HIV-1 and resistant mutant variants. Thymidine analogue mutations (TAMs) can increase the rates of phosphorolytic excision of incorporated zidovudine-monophosphate (AZT-MP)- and stavudine-monophosphate (d4T-MP); however, it remains to be seen whether misincorporated chain-terminators are removed with similar efficiency.
METHODS: We have purified wild-type HIV-1 RT and TAMs-containing mutant enzymes in order to study the efficiency of both incorporation and excision of mismatched AZT-MP and d4T-MP in gel-based assays.
RESULTS: Steady-state kinetic analyses revealed that wild-type HIV-1 RT is able to incorporate thymidineanalogues opposite template G with similar high efficiency as seen with the natural counterpart dTMP. In contrast, mismatch formation was hardly observed with lamivudine-MP (3TC-MP) or other non-thymidine analogues. We next studied whether misincorporated AZT-MP or d4T-MP can be excised in the presence of ATP, which can act as a pyrophosphate donor. We found that the wild-type enzyme was neither capable of removing AZT-MP, nor d4T-MP, while TAMs-containing mutant enzymes were able to remove AZT-MP. Moreover, our data also show that the M184V mutation, which has previously been associated with AZT resensitization effects, can diminish the efficiency of excision of G-mispaired AZT-MP when introduced in a background of TAMs. Most importantly, we found that the removal of mispaired d4T-MP was completely blocked, although different enzymes containing mutations at positions 41, 67, 70, 210, 215 and 219 clearly facilitated the excision of d4T-MP opposite the correct template base.
CONCLUSIONS: Our biochemical data suggest that thymidine analogue RT inhibitors can be misincorporated, provided that the concentration of the nucleoside triphosphate is sufficiently high. The finding that TAMs-containing mutant enzymes are unable to remove mispaired d4T-MP may help to explain why the increase in phenotypic resistance to d4T is only minimal when measured in cell-based assays.
PRESENTING AUTHOR: M Götte
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2004-06-08
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