Abstract
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 k(pol): 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 wild-type 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.
Highlights
Ited by the emergence of drug-resistant variants
We find that K65R and L74V do not combine into a ddN-superresistant virus and that the simultaneous presence of both substitutions in reverse transcriptase (RT) affects viral replication capacity through alteration of the interaction of natural nucleotides with the RT active site
We conclude that K65R and L74V are not synergistic in terms of resistance, that the inability to select and maintain the K65R/L74V virus is due to moderate resistance to ddNs combined with a loss of viral replication capacity, and that this loss of replication capacity is correlated to a decreased ability of RT to use natural nucleotides
Summary
Recombinant HIV-1 Molecular Clone Constructions—The 1258-bp ClaI-Eco47III fragment of the p66RTB vector containing mutations K65R, L74V, or K65R/L74V was introduced in the same restriction sites as described previously into the p66RTB-AD8 vector [20]. The reaction was performed by mixing a solution containing 50 nM (active sites) HIV-1 RT bound to 100 nM primer/template in RT buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.05% Triton X-100), and a variable concentration of dNTP in 6 mM MgCl2. The processivity assay was performed under the same experimental conditions with poly(rA)/oligo(dT) for 2 min with 2 g/l heparin and an excess of unlabeled primer/template added to the dTTP/MgCl2 mixture. The adenine at position 5 of the template was mutated to thymine, the counterpart of the incoming nucleotide analogue dATP and ddATP The conformation of this fifth template nucleotide and the side chain of selected amino acids 65 and 74 were minimized within the Biopolymer module of GenMol using a conformational analysis method. The incoming nucleotide was docked into the DNA-polymerase active site from the positioning of dTTP present in the x-ray structure ternary complex (nucleoside binding site, N site).
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