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Papers In Press, published online ahead of print March 23, 2004
Centre National de la Recherche Scientifique and Université Aix-Marseille I and II, UMR 6098, Marseille, Cedex 09 13288
Corresponding Author: bruno{at}afmb.cnrs-mrs.fr
Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2´,3´-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI while K65R is not. Here, we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5 fold) relative to wild-type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant kpol: 22-fold (from 7.2 to 0.33 s-1) for K65R RT and 84-fold (from 7.2 to 0.086 s-1) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is WT RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic, and might give a mechanism for a decreased viral fitness at the molecular level.
J. Biol. Chem, 10.1074/jbc.M313534200
Submitted on December 10, 2003
Revised on March 23, 2004
Accepted on March 23, 2004
A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions
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