HIV-1 drug resistance mutations are often inversely correlated with viral fitness, which remains poorly described at the molecular level. Some resistance mutations can also suppress resistance caused by other resistance mutations. We report the molecular mechanisms by which a virus resistant to lamivudine with the M184V reverse transcriptase mutation shows increased susceptibility to tenofovir and can suppress the effects of the tenofovir resistance mutation K65R. Additionally, we report how the decreased viral replication capacity of resistant viruses is directly linked to their decreased ability to use natural nucleotide substrates and that combination of the K65R and M184V resistance mutations leads to greater decreases in viral replication capacity. All together, these results define at the molecular level how nucleoside-resistant viruses can be driven to reduced viral fitness.
The amino acid change K65R in human immunodeficiency virus type 1-reverse transcriptase (RT) confers viral resistance to various 2,3-dideoxynucleoside drugs in vivo. Using pre-steady state kinetic methods, we found that K65R-reverse transcriptase is 3.2-14-fold resistant to 2,3-dideoxynucleotides in vitro relative to wild-type reverse transcriptase, in agreement with resistance levels observed in vivo. A decreased catalytic rate constant k pol mostly accounts for the lower incorporation efficiency observed for 2,3-dideoxynucleotides. Examination of the crystal structure of the RT⅐DNA⅐dNTP complex suggested that both the charge at position 65 and the 3-OH of the incoming nucleotide act in synergy during the creation of the phosphodiester bond, resulting in a more pronounced decreased catalytic rate constant for 2,3-dideoxynucleotides than for dNTPs. This type of intramolecular activation of the leaving phosphate by the 3-OH group appears to be conserved in several nucleotide phosphotransferases. These data were used to design dideoxynucleotide analogues targeting K65R RT specifically. ␣-Boranophosphate ddATP was found to be a 2-fold better substrate than dATP and inhibited DNA synthesis by K65R RT 153-fold better than ddATP. This complete suppression of drug resistance at the nucleotide level could serve for other reverse transcriptases for which drug resistance is achieved at the catalytic step.
Nucleoside analogues are currently used to treat human immunodeficiency virus infections. The appearance of up to five substitutions (A62V, V75I, F77L, F116Y, and Q151M) in the viral reverse transcriptase promotes resistance to these drugs, and reduces efficiency of the antiretroviral chemotherapy. Using pre-steady state kinetics, we show that Q151M and A62V/V75I/F77L/F116Y/ Q151M substitutions confer to reverse transcriptase (RT) the ability to discriminate an analogue relative to its natural counterpart, and have no effect on repair of the analogue-terminated DNA primer. Discrimination results from a selective decrease of the catalytic rate constant k pol : 18-fold (from 7 to 0.3 s ؊1 ), 13-fold (from 1.9 to 0.14 s ؊1 ), and 12-fold (from 13 to 1 s ؊1 ) in the case of ddATP, ddCTP, and 3-azido-3-deoxythymidine 5-triphosphate (AZTTP), respectively. The binding affinities of the triphosphate analogues for RT remain unchanged. Molecular modeling explains drug resistance by a selective loss of electrostatic interactions between the analogue and RT. Resistance was overcome using ␣-boranophosphate nucleotide analogues. Using A62V/V75I/ F77L/F116Y/Q151M RT, k pol increases up to 70-and 13-fold using ␣-boranophosphate-ddATP and ␣-boranophosphate AZTTP, respectively. These results highlight the general capacity of such analogues to circumvent multidrug resistance when RT-mediated nucleotide resistance originates from the selective decrease of the catalytic rate constant k pol . The human immunodeficiency virus (HIV)1 infects more than 40 million individuals in the world. 3Ј-Azido-3Ј-deoxythymidine (AZT, zidovudine) was the first antiretroviral drug to receive approval from the FDA in 1987 to treat HIV-1-infected patients. AZT is a nucleoside analogue acting on viral replication. It is metabolically activated by cellular kinases of the host cell to its corresponding triphosphate form AZTTP before reaching its target, reverse transcriptase (RT). RT is an essential viral DNA polymerase responsible for viral DNA synthesis. AZTTP is a poor substrate for cellular DNA polymerases, but is incorporated into the nascent viral DNA strand by RT with the same efficiency as its natural nucleotide counterpart dTTP. Because AZT lacks a 3Ј-hydroxyl group (3Ј-OH) on its ribose moiety, AZTMP is incorporated into DNA and viral DNA synthesis is terminated.The prolonged use of AZT as the sole drug in the clinic has resulted in the emergence of AZT-resistant viruses (1). A set of six specific substitutions on RT (M41L, D67N, K70R, T215Y or F, L210W, and K219E or Q) gives rise to high level AZT resistance (2), the appearance of T215F or Y being the most important substitution. A long awaited mechanism of AZT resistance because of these mutations has been proposed, based on biochemical studies using purified reverse transcriptase: AZTresistant RT is able to catalyze a primer-unblocking reaction related to pyrophosphorolysis (3, 4) to remove the chain-terminating AZTMP. This "repair" reaction allows the RT to resume elongation of the primer DNA.
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