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Application of a colorimetric chain‐termination assay for characterization of reverse transcriptase from 3′‐azido‐2′,3′‐deoxythymidine‐resistant HIV isolates
Author(s) -
Shao XingWu,
Hjalmarsson Sandra,
Lennerstrand Johan,
Svennerholm Bo,
Blomberg Jonas,
Källander Clas F. R.,
Gronowitz J. Simon
Publication year - 2002
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.2002.tb01184.x
Subject(s) - reverse transcriptase , zidovudine , primer (cosmetics) , enzyme , microbiology and biotechnology , biology , nucleotidyltransferase , virology , polymerase chain reaction , nucleotide , virus , chemistry , gene , rna , biochemistry , viral disease , organic chemistry
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, P <0.01). The second assay, which was designed specifically for measurement of chain termination in the absence of dNTP substrate (as the concentration of AZT‐TP giving 50% residual primer function, or CT 50 ), revealed a more than 600‐fold difference between the different isolate RTs. For the majority of enzymes there was a strict correlation between the results from the two assays; however, four isolates exhibited significantly higher CT 50 /IC 50 ratios than the other isolates. These differences were not related to sensitivity of the corresponding viruses to AZT but to the occurrence of certain mutations in their pol gene. The four deviating isolates contained either a minimum of four AZT‐specific substitutions, including Thr‐215→Tyr (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.