Inhibition of the initiation of HIV-1 reverse transcription by 3 '-azido-3'-deoxythymidine - Comparison with elongation

Initiation of human immunodeficiency virus-1 reverse transcription requires formation of a complex containing the viral RNA, primer tRNA(3)(Lys), and reverse transcriptase, Initiation, corresponding to addition of the first s ix nucleotides to tRNA(3)(Lys), is distinguished from elongation by its hig h specificity and low efficiency (processivity), Here, we compared the inhi bition of initiation and elongation of reverse transcription by 3'-azido-3' -deoxythymidine 5'-triphosphate (AZTTP), the active form of 3'-azido-3'-deo xythymidine. We report the first detailed study of nucleotide binding, disc rimination, and pyrophosphorolysis by the authentic initiation complex, We showed that the initiation and elongation complexes bound AZTTP and dTTP wi th the same affinity, while the polymerization rates were reduced by 148-16 0-fold during initiation. The pyrophosphorolysis rate of dTTP was reduced b y the same extent, indicating that the polymerization equilibrium is the sa me in the two phases. The efficient unblocking of the 3'-azido-3'-deoxythym idine 5'-monophosphate (AZTMP)-terminated primer by pyrophosphorolysis sign ificantly relieved inhibition of DNA synthesis during elongation in the pre sence of physiological pyrophosphate concentrations. Remarkably, although p yrophosphorolysis of dTMP and AZTMP were equally efficient during elongatio n, reverse transcriptase was almost totally unable to unblock the AZTMP-ter minated primer during initiation. As a result, inhibition of reverse transc ription by AZTTP was more efficient during initiation than elongation of re verse transcription, despite a reduced selectivity of incorporation.