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Biotechnology and Applied Biochemistry (2002) 35, (155–164) (Printed in Great Britain)

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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

Xing-Wu Shao*†¹, Sandra Hjalmarsson‡, Johan Lennerstrand§, Bo Svennerholm¶, Jonas Blomberg‡, Clas F. R. Källander† 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, Göteborg University, Göteborg, Sweden

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; CT₅₀, 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; ED₅₀, AZT dose giving 50% reduction of viral replication; BrdUTP, 5-bromodeoxyuridine 5´-triphosphate; BrdU, 5-bromodeoxyuridine.

¹To whom correspondence should be addressed, at Cavidi Tech AB (e-mail xingwu@cavidi.se).

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 IC₅₀), gave an approx. 9-fold variation in sensitivity to AZT-TP. There was a correlation between the IC₅₀ 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 CT₅₀), 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 CT₅₀/IC₅₀ 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.

Received 23 May 2001/28 January 2002; accepted 4 February 2002

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