Single-step kinetics of HIV-1 reverse transcriptase mutants responsible for virus resistance to nucleoside inhibitors zidovudine and 3-TC

Biochemistry. 1997 Aug 19;36(33):10292-300. doi: 10.1021/bi970512z.


Two mutants of HIV-1 reverse transcriptase (RT) associated with high-level resistance of the virus to AZT (RT-AZT: D67N, K70R, T215Y, K219Q, and M41L) or 3-TC (RT-3TC: M184V) were expressed in Escherichia coli and purified. None of these mutants showed significant changes in the affinity and kinetics of binding to a DNA/DNA primer/template. RT-AZT was investigated in detail with respect to its kinetics of incorporation of nucleotides. No change in the relative rates of TMP and AZTMP incorporation could be detected for RT-AZT with respect to wild type RT. These results imply that there is no increased discrimination against AZTTP in the mutant. This was found for DNA/DNA and DNA/RNA primer/template. Additionally, rapid kinetics of incorporation of 3'-amino-3'-deoxythymidine 5'-monophosphate (a possible metabolite of AZT) were investigated and compared with TMP incorporation, but no difference in its relative rates of incorporation between wild type RT and RT-AZT was detected. In contrast, the already very slow rate of incorporation of 3-TCMP seen with wild type enzyme was drastically reduced (by a factor of 23 and 36 with DNA/DNA primer/template and DNA/RNA primer/template, respectively) for RT-3TC, showing a clear correlation between in vitro and in vivo effects. The affinity of 3-TCTP to the RT-3TC-primer/template complex was not affected by the mutation M184V. A 1.6-fold cross-resistance to ddATP, the converted form of the prodrug ddI, could also be shown for RT-3TC, but no cross-resistance to ddCTP was detected. Additionally, rapid kinetics of AZTMP incorporation by RT-3TC were investigated. There was an indication of a slightly higher rate of incorporation of AZTMP by RT-3TC than wild type RT.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-HIV Agents / pharmacology
  • Drug Resistance, Microbial / genetics
  • HIV Reverse Transcriptase / genetics
  • HIV Reverse Transcriptase / metabolism*
  • HIV-1 / drug effects*
  • HIV-1 / enzymology
  • Kinetics
  • Lamivudine / pharmacology*
  • Mutation
  • Reverse Transcriptase Inhibitors / pharmacology*
  • Zidovudine / pharmacology*


  • Anti-HIV Agents
  • Reverse Transcriptase Inhibitors
  • Lamivudine
  • Zidovudine
  • HIV Reverse Transcriptase