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 six nucleotides to tRNA(3)(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.
Rigourd
M
M
UPR 9002 du CNRS, IBMC, 15 rue René Descartes, 67084 Strasbourg cedex, France.
Lanchy
J M
JM
Le Grice
S F
SF
Ehresmann
B
B
Ehresmann
C
C
Marquet
R
R
D001483:Base Sequence; D017931:DNA Primers; D054306:Dideoxynucleotides; D011756:Diphosphates / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D013942:Thymine Nucleotides / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D015215:Zidovudine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*
D017931:DNA Primers; D054306:Dideoxynucleotides; D011756:Diphosphates; D013942:Thymine Nucleotides; D015215:Zidovudine; C052873:zidovudine triphosphate; D054303:HIV Reverse Transcriptase; C024157:thymidine 5'-triphosphate