Application of a Chain Termination Assay for Characterization of Reverse Transcriptase from AZT-Resistant HIV Isolates

Abstract

An enzymatic assay based on utilization of one primer/enzyme molecule was specifically designed for evaluation of the chain termination capacity of reverse transcriptase (RT) from HIV-1 isolates. In this assay system (CT assay) there was a 3.2-fold difference between the AZT-triphosphate (AZT-TP) concentrations required to terminate 50% of the primers (TC50) for a highly resistant isolate, carrying the four common mutations at positions 67, 70, 215, and 219; and two wild type isolates. Two of three other isolates with reduced sensitivity to AZT in cell culture exhibited intermediate values in CT assay, while one behaved as the wild type isolates. There was a correlation P = 0.05 ( r = 0.86, n = 6) between the ED50 values found in cell culture and the TC50 values found in CT assay. This relationship was not found in a similar assay system which measured competition between AZT-TP and 1 μM tymidine triphosphate (TTP) at the enzymatic level. The sequence data of the current isolates gave some information concerning which mutations in the RT gene specifically affect the enzymatic properties measured in CT assay. Mutation only at amino acid 70 had no effect, but the TC50 values found increased with accumulation of the other common AZT resistance mutations.