Biotechnol. Appl. Biochem. (2002) 35, 155–164 (Printed in Great Britain) 155 Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3-azido-2,3- deoxythymidine-resistant HIV isolates Xing-Wu Shao*† 1 , Sandra Hjalmarsson‡, Johan Lennerstrand§, Bo Svennerholm¶, Jonas Blomberg‡, Clas F. R. Ka  llander † and J. Simon Gronowitz† *MTC , Karolinska Institute, Stockholm, Sweden, †Cavidi Tech AB, Uppsala Science Park, S-751 83 Uppsala, Sweden, ‡Department of Clinical Virology, Uppsala University, Uppsala, Sweden, §Division of Clinical Virology, Huddinge University Hospital, Stockholm, Sweden, and ¶Department of Clinical Virology, Go  teborg University, Go  teborg, Sweden Two different enzyme assays, both based on the in- teraction 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 monophos- phate 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 sub- stitution has previously been recorded in connection with AZT/Foscarnet combination therapy. Introduction For the last decade 3-azido-2,3-deoxythymidine (AZT) has been used extensively in the treatment of HIV infection. The drug is a thymidine analogue that is phosphorylated intracellularly to AZT triphosphate (AZT-TP) by cellular enzymes of the thymidine salvage pathway. AZT-TP is used as an alternative substrate by the viral reverse transcriptase (RT) and terminates the DNA chain elongation as it lacks a 3-hydroxyl group [1]. Prolonged therapy with AZT com- monly leads to the development of resistant virus. This process is associated with the gradual appearance of mutations in the viral pol gene, each leading to defined amino acid substitutions. For example, changes at codons 41, 67, 70, 210, 215 and 219 result in resistance to AZT [2–7]. The corresponding mutant virus has been shown to be up to several hundred times less sensitive to AZT inhibition in cell culture compared with wild-type isolates. However, the biochemical mechanism of resistance conferred by these mutations has remained a puzzle for a long period. This is because only minor or no differences were revealed when comparing the kinetic properties of mutant enzymes with wild type in the presence of different AZT-TP concen- trations [4,5,8–11]. On the other hand, therapy with multiple dideoxy nucleosides results in the appearance of another set of mutations, the Glu-151  Met complex, that is associated with high-level resistance to multiple dideoxy nucleosides. In this case, a decreased sensitivity to AZT in cell culture was mirrored by a corresponding increase in the kinetic con- stants for AZT-TP inhibition at the enzymic level [12]. During the last few years a possible biochemical explanation has emerged. Arion et al. [13] showed that enhanced removal of AZT monophosphate (AZT-MP) from terminated primers could occur for an AZT-resistant mutant compared with wild-type RT, and this was due to pyrophos- phate-dependent pyrophosphorolysis, the reverse reaction Key words : drug sensitivity, enzyme assay, phenotype assay, primer rescue, pyrophosphorolysis reaction. Abbreviations used : AZT, 3-azido-2,3-deoxythymidine ; AZT-MP, AZT monophosphate ; AZT-TP, AZT triphosphate ; CT 50 , concentration of AZT-TP giving 50 % residual primer function ; SIV, simian immunodeficiency virus ; PBMC, peripheral blood mononuclear cell ; RT, reverse transcriptase ; CT assay, chain-termination assay ; ED 50 , AZT dose giving 50 % reduction of viral replication ; BrdUTP, 5-bromodeoxyuridine 5-triphosphate ; BrdU, 5-bromodeoxyuridine. 1 To whom correspondence should be addressed, at Cavidi Tech AB (e-mail xingwucavidi.se).  2002 Portland Press Ltd