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

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.The efficacy of current anti-HIV 1 treatments with nucleoside analogues (nucleoside reverse transcriptase inhibitors) is limited by the emergence of drug-resistant variants. Because antiretroviral therapy does not completely suppress viral replication, a resulting selection pressure favors the emergence of mutant viruses that partially or completely lose sensitivity to one or several inhibitors (1, 2). Such an example is given by the progressive appearance of primary mutations in the viral pol gene encoding the reverse transcriptase (RT) after prolonged treatments that make use of dideoxynucleosides. K65R and L74V substitutions in RT are typically associated with a moderate resistance to ddI and ddC, and these substitutions are directly responsible for a loss of efficacy of these drugs in patients carrying the corresponding mutant viruses.We previously reported the molecular role of the K65R substitution in the multiple resistance to dideoxynucleotides (3). Lysine at position 65 is located in the flexible ␤3-␤4 loop located in the finger domain of the p66 monomer of RT. The side chain of the Arg-65 interacts with the ␥-phosphate of the incoming nucleotide and alters the catalytic rate of creation of the phosphodiester bond for dideoxynucleotides specifically (3). The K65R mutation is found infrequently (less than 2%) on viral isolates from patients treated with ddI and ddC and gives a moderate 2-10-fold decrease in the sensitivity to these two drugs (4, 5). K65R also has been described to give in vitro RT-media...

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

“…For example, the M184V and V75T mutations in HIV-1 RT increase the NRTI-TP K d without significantly impacting the k pol (9,10,26). Other mutations, such as Q151M and L74V, decrease the k pol without affecting the K d (7,8). Consistent with prior reports, our data show that K65R predominantly exerts its discrimination effects by selectively decreasing the k pol (9,11,27), although small changes in K d were noted in this study and others (14,27).…”

Kinetics of Inhibition of HIV Type 1 Reverse Transcriptase-Bearing NRTI-associated Mutations by Apricitabine Triphosphate

“…The K65R mutation has been shown to use both mechanisms, depending on the NRTI-TP studied (Deval et al, 2004b;Deval et al, 2004a;White et al, 2005). We studied the binding/incorporation of apricitabine-TP for WT and relevant mutant RTs by determining the ratio of the inhibition constant (K i ) for apricitabine-TP to the K m for its natural counterpart dCTP.…”

“…The K65R mutation has been shown to decrease the binding or incorporation of zidovudine-TP as well as the k pol of tenofovir diphosphate compared with WT RT, with only a modest effect on K d (Deval et al, 2004a;White et al, 2005). Apricitabine-TP has been shown to inhibit HIV-1 WT RT with a K i that is twofold lower than that of lamivudine-TP in vitro .…”

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…It has been shown that L74V in the context of the NL4-3 laboratory strain has an 11% loss of fitness compared to that of the wild type or K70R (154). Biochemical studies have shown that this replication decrease is associated with a reduction in the incorporation of nucleotides compared to the wild type and a decrease in the initiation of minus-strand and plus-strand DNA synthesis from the tRNA 3 Lys and polypurine tract primers (47,49). A reduced ability of L74V viruses to synthesize proviral DNA was demonstrated in cell culture (61).…”

“…L74V and K65R, which are rarely found together in clinical isolates, have markedly antagonistic effects on viral fitness that correlate with reductions in the ability to utilize normal nucleotides during DNA synthesis (47). Similarly, K65R and M184V also have antagonistic effects on fitness that are associated with abnormalities in nucleotide incorporation (46).…”