TY - JOUR
T1 - Inhibition of the initiation of HIV-1 reverse transcription by 3'-azido-3'-deoxythymidine
T2 - Comparison with elongation
AU - Rigourd, Mickaël
AU - Lanchy, Jean Marc
AU - Le Grice, Stuart F.J.
AU - Ehresmann, Bernard
AU - Ehresmann, Chantal
AU - Marquet, Roland
PY - 2000/9/1
Y1 - 2000/9/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0034282644&partnerID=8YFLogxK
U2 - 10.1074/jbc.M003262200
DO - 10.1074/jbc.M003262200
M3 - Article
C2 - 10864929
AN - SCOPUS:0034282644
SN - 0021-9258
VL - 275
SP - 26944
EP - 26951
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 35
ER -