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

Mickaël Rigourd, Jean Marc Lanchy, Stuart F.J. Le Grice, Bernard Ehresmann, Chantal Ehresmann, Roland Marquet

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Initiation of human immunodeficiency virus-1 reverse transcription requires formation of a complex containing the viral RNA, primer tRNA3/(Lys), and reverse transcriptase. Initiation, corresponding to addition of the first six nucleotides to tRNA3/(Lys), is distinguished from elongation by its high specificity and low efficiency (processivity). Here, we compared the inhibition of initiation and elongation of reverse transcription by 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), the active form of 3'-azido-3'-deoxythymidine. We report the first detailed study of nucleotide binding, discrimination, and pyrophosphorolysis by the authentic initiation complex. We showed that the initiation and elongation complexes bound AZTTP and dTTP with the same affinity, while the polymerization rates were reduced by 148-160-fold during initiation. The pyrophosphorolysis rate of dTTP was reduced by the same extent, indicating that the polymerization equilibrium is the same in the two phases. The efficient unblocking of the 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated primer by pyrophosphorolysis significantly relieved inhibition of DNA synthesis during elongation in the presence of physiological pyrophosphate concentrations. Remarkably, although pyrophosphorolysis of dTMP and AZTMP were equally efficient during elongation, reverse transcriptase was almost totally unable to unblock the AZTMP-terminated primer during initiation. As a result, inhibition of reverse transcription by AZTTP was more efficient during initiation than elongation of reverse transcription, despite a reduced selectivity of incorporation.

Original languageEnglish
Pages (from-to)26944-26951
Number of pages8
JournalJournal of Biological Chemistry
Issue number35
StatePublished - Sep 1 2000


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