Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3'-azido-2', 3'-deoxythymidine-resistant HIV isolates

Two different enzyme assays, both based on the interaction of native reverse transcriptase (RT) and 3'-azido-2',3'-deoxythymidine triphosphate (AZT-TP), were used to characterize the enzymes from 18 HIV-1 isolates with decreased sensitivity to AZT in cell culture. The first assay, whi...

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Bibliographic Details
Published in:Biotechnology and applied biochemistry 2002-06, Vol.35 (Pt 3), p.155-164
Main Authors: SHAO, Xing-Wu, HJALMARSSON, Sandra, LENNERSTRAND, Johan, SVENNERHOLM, Bo, BLOMBERG, Jonas, KÄLLANDER, Clas F. R, GRONOWITZ, J. Simon
Format: Article
Language:English
Subjects:
Anti-HIV Agents - administration & dosage
Biological and medical sciences
Biotechnology
Cells, Cultured
Colorimetry - methods
Dideoxynucleotides
Dose-Response Relationship, Drug
Drug Resistance, Viral - physiology
Enzyme-Linked Immunosorbent Assay - methods
Fundamental and applied biological sciences. Psychology
HIV-1 - drug effects
HIV-1 - enzymology
Humans
Leukocytes, Mononuclear - virology
Methods. Procedures. Technologies
Others
Reverse Transcriptase Inhibitors - administration & dosage
RNA-Directed DNA Polymerase - metabolism
Thymine Nucleotides - administration & dosage
Various methods and equipments
Zidovudine - administration & dosage
Zidovudine - analogs & derivatives
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Summary:Two different enzyme assays, both based on the interaction of native reverse transcriptase (RT) and 3'-azido-2',3'-deoxythymidine triphosphate (AZT-TP), were used to characterize the enzymes from 18 HIV-1 isolates with decreased sensitivity to AZT in cell culture. The first assay, which measures the balance between incorporation and excision of AZT monophosphate in the presence of dNTP substrate (in terms of IC(50)), gave an approx. 9-fold variation in sensitivity to AZT-TP. There was a correlation between the IC(50) values and the sensitivity of the corresponding virus to AZT in cell culture (r=0.60, PTyr (isolates 134 and 143), or some of the known specific substitutions combined with Thr-39-->Ala (isolates 80 and 157). The Thr-39-->Ala substitution has previously been recorded in connection with AZT/Foscarnet combination therapy.
ISSN:0885-4513
1470-8744
1470-8744
DOI:10.1042/BA20010031