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Single-Step Kinetics of HIV-1 Reverse Transcriptase Mutants Responsible for Virus Resistance to Nucleoside Inhibitors Zidovudine and 3-TC

Title data

Krebs, Ruth ; Immendörfer, Ulrike ; Thrall, Sara H. ; Wöhrl, Birgitta M. ; Goody, Roger S.:
Single-Step Kinetics of HIV-1 Reverse Transcriptase Mutants Responsible for Virus Resistance to Nucleoside Inhibitors Zidovudine and 3-TC.
In: Biochemistry. Vol. 36 (1997) Issue 33 . - pp. 10292-10300.
ISSN 0006-2960
DOI: https://doi.org/10.1021/bi970512z

Abstract in another language

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.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.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers > Lehrstuhl Biopolymere - Apl. Prof. Dr. Birgitta Wöhrl
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 29 May 2019 10:19
Last Modified: 29 May 2019 10:19
URI: https://eref.uni-bayreuth.de/id/eprint/49146