ABSTRACT:
Prolonged clinical use of nucleoside analogue chain terminators, such as zidovudine and lamivudine, gives rise to resistant viruses that contain mutations in the reverse transcriptase (RT) enzyme. The mutant of HIV-1 RT associated with high-level resistance to lamivudine (RT M184V) shows diminished incorporation of the chain terminator. in contrast, mutant enzymes associated with zidovudine resistance, for example RT D67N/K70R, show an increased rate of pyrophosphorolysis rather than diminished tolerance of the nucleoside analogue. This indicates that chain termination is not necessarily an irreversible step and, consequently, both a decreased forward reaction as well as an increased reverse reaction (pyrophosphorolysis), with respect to a given nucleoside analogue, can contribute to higher levels of drug resistance. We have developed cell-free assays to specifically monitor incorporation and pyrophosphorolytic cleavages of zidovudine and lamivudine during the initiation of HIV-1 plus-strand DNA synthesis.
These reactions employ primer-template systems that correspond to the polypurine tract (PPT) and use either wild-type or mutant enzymes to investigate the mechanisms of resistance to both drugs. We have demonstrated that zidovudine-TP is efficiently incorporated with both wild-type RT and RT D67N/K70R. However, the mutant enzyme showed significantly increased sensitivity towards pyrophosphate that resulted in rescue of DNA synthesis. In contrast, lamivudine-TP was moderately accepted by wild-type RT and much decreased rates of incorporation were seen with RT M184V. Most importantly, our data indicate that the reverse reaction followed the same order. Pyrophosphorolysis by wild-type RT was less pronounced when lamivudine-TP was incorporated and the mutant enzyme showed a strongly diminished back reaction at physiological pyrophosphate levels. Moreover, the 184V mutation also severely compromises pyrophosphorolytic cleavages of zidovudine-terminated primers.
Thus, rescue of DNA synthesis, terminated with zidovudine or lamivudine, is impaired by HIV resistance to lamivudine. These data are consistent with tissue culture and clinical data, indicating that the 184V mutation caused an increase in zidovudine sensitivity in the context of viruses resistant to zidovudine and zidovudine/lamivudine combination treatment.
