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Effects of specific zidovudine resistance mutations and substrate structure on nucleotide-dependent primer unblocking by human immunodeficiency virus type 1 reverse transcriptase.

ABSTRACT: Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3'-azido-3'-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that pi-pi interactions between RT and an aromatic structure may be facilitated by these mutations.

SUBMITTER: Meyer PR

PROVIDER: S-EPMC127181 | biostudies-literature | 2002 May

REPOSITORIES: biostudies-literature

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Effects of specific zidovudine resistance mutations and substrate structure on nucleotide-dependent primer unblocking by human immunodeficiency virus type 1 reverse transcriptase.

Meyer Peter R PR Matsuura Suzanne E SE Tolun Adviye A AA Pfeifer Ingrid I So Antero G AG Mellors John W JW Scott Walter A WA

Antimicrobial agents and chemotherapy 20020501 5

Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3'-azido-3'-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was d ...[more]

PMID: 11959594

Similar Datasets

Drug resistance mutations in the nucleotide binding pocket of human immunodeficiency virus type 1 reverse transcriptase differentially affect the phosphorolysis-dependent primer unblocking activity in the presence of stavudine and zidovudine and its inhibition by efavirenz.

Project description:Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) derivatives with D113E, Y115F, F116Y, Q151E/N, and M184V mutations were studied for their phosphorolysis-mediated resistance to the nucleoside RT inhibitors (NRTIs) zidovudine and stavudine and for their inhibition by the nonnucleoside analogs (NNRTIs) efavirenz and nevirapine. The results presented here indicate that these single amino acid substitutions within the nucleotide binding pocket of the viral RT can independently affect different enzymatic properties, such as catalytic efficiency, drug binding, and phosphorolytic activity. Moreover, small local alterations of the physicochemical properties of the microenvironment around the active site can have profound effects on some NRTIs while hardly affecting other ones. In conclusion, even though different mutations within the nucleotide binding pocket of HIV-1 RT can result in a common phenotype (i.e., drug resistance), the molecular mechanisms underlying this phenotype can be very different. Moreover, the same mutation can give rise to different phenotypes depending on the nature of the substrates and/or inhibitors.

| S-EPMC538890 | biostudies-literature

Relationship between 3'-azido-3'-deoxythymidine resistance and primer unblocking activity in foscarnet-resistant mutants of human immunodeficiency virus type 1 reverse transcriptase.

Project description:Phosphonoformate (foscarnet) is a pyrophosphate (PP(i)) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PP(i) binding site on the enzyme. HIV-1 RT can unblock a chain-terminated DNA primer by phosphorolytic transfer of the terminal residue to an acceptor substrate (PP(i) or a nucleotide such as ATP) which also interacts with the PP(i) binding site. Primer-unblocking activity is increased in mutants of HIV-1 that are resistant to the chain-terminating nucleoside inhibitor 3'-azido-3'-deoxythymidine (AZT). We have compared the primer-unblocking activity for HIV-1 RT containing various foscarnet resistance mutations (K65R, W88G, W88S, E89K, S117T, Q161L, M164I, and the double mutant Q161L/H208Y) alone or in combination with AZT resistance mutations. The level of primer-unblocking activity varied over a 150-fold range for these enzymes and was inversely correlated with foscarnet resistance and directly correlated with AZT resistance. Based on published crystal structures of HIV-1 RT, many of the foscarnet resistance mutations affect residues that do not make direct contact with the catalytic residues of RT, the incoming deoxynucleoside triphosphate (dNTP), or the primer-template. These mutations may confer foscarnet resistance and reduce primer unblocking by indirectly decreasing the binding and retention of foscarnet, PP(i), and ATP. Alternatively, the binding position or orientation of PP(i), ATP, or the primer-template may be changed in the mutant enzyme complex so that molecular interactions required for the unblocking reaction are impaired while dNTP binding and incorporation are not.

| S-EPMC155000 | biostudies-literature

Coresistance to zidovudine and foscarnet is associated with multiple mutations in the human immunodeficiency virus type 1 reverse transcriptase.

Project description:Human immunodeficiency virus type 1 (HIV-1) isolates obtained from a patient with AIDS were assessed for coresistance to foscarnet and zidovudine. An HIV-1 strain (AP20) coresistant to foscarnet and zidovudine was isolated after 20 months of continuous combination therapy. The reverse transcriptase (RT) gene of AP20 had 41 substitutions which were different from the HXB2-D sequence and 9 that were different from the sequence of its foscarnet-sensitive, zidovudine-resistant progenitor virus (AP6). Six of these mutations were nonpolymorphic (T39A, V108I, K166R, K219R, K223Q, and L228R). Both strains had the conventional mutations mediating zidovudine resistance. In vivo selection may result in HIV-1 strains that are coresistant to foscarnet and zidovudine, but coresistance appears to require a complex evolutionary path and multiple RT mutations.

| S-EPMC105992 | biostudies-literature

Hypersusceptibility to substrate analogs conferred by mutations in human immunodeficiency virus type 1 reverse transcriptase.

Project description:Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed "random scanning mutagenesis" of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.

| S-EPMC1489025 | biostudies-literature

Mutations M184V and Y115F in HIV-1 reverse transcriptase discriminate against "nucleotide-competing reverse transcriptase inhibitors".

Project description:Indolopyridones are potent inhibitors of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1). Although the structure of these compounds differs from established nucleoside analogue RT inhibitors (NRTIs), previous studies suggest that the prototype compound INDOPY-1 may bind in close proximity to the polymerase active site. NRTI-associated mutations that are clustered around the active site confer decreased, e.g. M184V and Y115F, or increased, e.g. K65R, susceptibility to INDOPY-1. Here we have studied the underlying biochemical mechanism. RT enzymes containing the isolated mutations M184V and Y115F cause 2-3-fold increases in IC(50) values, while the combination of the two mutations causes a >15-fold increase. K65R can partially counteract these effects. Binding studies revealed that the M184V change reduces the affinity to INDOPY-1, while Y115F facilitates binding of the natural nucleotide substrate and the combined effects enhance the ability of the enzyme to discriminate against the inhibitor. Studies with other strategic mutations at residues Phe-61 and Ala-62, as well as the use of chemically modified templates shed further light on the putative binding site of the inhibitor and ternary complex formation. An abasic site residue at position n, i.e. opposite the 3'-end of the primer, prevents binding of INDOPY-1, while an abasic site at the adjacent position n+1 has no effect. Collectively, our findings provide strong evidence to suggest that INDOPY-1 can compete with natural deoxynucleoside triphosphates (dNTPs). We therefore propose to refer to members of this class of compounds as "nucleotide-competing RT inhibitors" (NcRTIs).

| S-EPMC2573056 | biostudies-literature

Analysis of the Zidovudine Resistance Mutations T215Y, M41L, and L210W in HIV-1 Reverse Transcriptase.

Project description:Although anti-human immunodeficiency virus type 1 (HIV-1) therapies have become more sophisticated and more effective, drug resistance continues to be a major problem. Zidovudine (azidothymidine; AZT) was the first nucleoside reverse transcriptase (RT) inhibitor (NRTI) approved for the treatment of HIV-1 infections and is still being used, particularly in the developing world. This drug targets the conversion of single-stranded RNA to double-stranded DNA by HIV-1 RT. However, resistance to the drug quickly appeared both in viruses replicating in cells in culture and in patients undergoing AZT monotherapy. The primary resistance pathway selects for mutations of T215 that change the threonine to either a tyrosine or a phenylalanine (T215Y/F); this resistance pathway involves an ATP-dependent excision mechanism. The pseudo-sugar ring of AZT lacks a 3' OH; RT incorporates AZT monophosphate (AZTMP), which blocks the end of the viral DNA primer. AZT-resistant forms of HIV-1 RT use ATP in an excision reaction to unblock the 3' end of the primer strand, allowing its extension by RT. The T215Y AZT resistance mutation is often accompanied by two other mutations, M41L and L210W. In this study, the roles of these mutations, in combination with T215Y, were examined to determine whether they affect polymerization and excision by HIV-1 RT. The M41L mutation appears to help restore the DNA polymerization activity of RT containing the T215Y mutation and also enhances AZTMP excision. The L210W mutation plays a similar role, but it enhances excision by RTs that carry the T215Y mutation when ATP is present at a low concentration.

| S-EPMC4649230 | biostudies-literature

Variations in reverse transcriptase and RNase H domain mutations in human immunodeficiency virus type 1 clinical isolates are associated with divergent phenotypic resistance to zidovudine.

Project description:Mutations in the RNase H domain of human immunodeficiency virus type 1 RT have been reported to cause resistance to zidovudine (ZDV) in vitro. However, very limited data on the in vivo relevance of these mutations in patients exist to date. This study was designed to determine the relationship between mutations in the RNase H domain and viral susceptibility to nucleoside analogues. Viruses harboring complex thymidine analogue mutation (TAM) and nucleoside analogue mutation (NAM) profiles were evaluated for their phenotypic susceptibilities to ZDV, tenofovir (TNF), and the nonapproved nucleoside reverse transcriptase inhibitors (NRTIs) beta-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (Reverset), beta-D-5-fluorodioxolane-cytosine, and apricitabine. As controls, viruses from NRTI-naïve patients were also studied. The pol RT region (codons 21 to 250) of the viruses were sequenced and evaluated for mutations in the RNase H domain (codons 441 to 560) and the connection domain (codons 289 to 400). The results showed that viruses from patients failing multiple NRTI-containing regimens had distinct TAM and NAM profiles that conferred various degrees of resistance to ZDV (0.9- to >300-fold). Sequencing of the RNase H domain identified five positions (positions 460,468, 483, 512, and 519) at which extensive amino acid polymorphisms common in both wild-type viruses and viruses from treated patients were identified. No mutations were observed at positions 539 and 549, which have previously been associated with ZDV resistance. Mutations in the RNase H domain did not appear to correlate with the levels of phenotypic resistance to ZDV. Although some mutations were also observed in the connection domain, the simultaneous presence of the L74V and M184V mutations was the most significant determinant of phenotypic resistance to ZDV in patients infected with viruses with TAMs.

| S-EPMC2151428 | biostudies-literature

Exploring the Reaction Mechanism of HIV Reverse Transcriptase with a Nucleotide Substrate.

Project description:Enzymatic reactions consist of several steps: (i) a weak binding event of the substrate to the enzyme, (ii) an induced fit or a protein conformational transition upon ligand binding, (iii) the chemical reaction, and (iv) the release of the product. Here we focus on step iii of the reaction of a DNA polymerase, HIV RT, with a nucleotide. We determine the rate and the free energy profile for the addition of a nucleotide to a DNA strand using a combination of a QM/MM model, the string method, and exact Milestoning. The barrier height and the time scale of the reaction are consistent with experiment. We show that the observables (free energies and mean first passage time) converge rapidly, as a function of the Milestoning iteration number. We also consider the substitution of an oxygen of the incoming nucleotide by a nonbridging sulfur atom and its impact on the enzymatic reaction. This substitution has been suggested in the past as a tool to examine the influence of the chemical step on the overall rate. Our joint computational and experimental study suggests that the impact of the substitution is small. Computationally, the differences between the two are within the estimated error bars. Experiments suggest a small difference. Finally, we examine step i, the weak binding of the nucleotide to the protein surface. We suggest that this step has only a small contribution to the selectivity of the enzyme. Comments are made on the impact of these steps on the overall mechanism.

| S-EPMC7260111 | biostudies-literature

The K101P and K103R/V179D mutations in human immunodeficiency virus type 1 reverse transcriptase confer resistance to nonnucleoside reverse transcriptase inhibitors.

Project description:Genotypic patterns associated with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in the absence of well-characterized resistance mutations were identified using a database (n > 47,000) of phenotype-genotype data. Among samples with no known NNRTI mutations, the most resistant samples contained K101P (n = 35) or a combination of K103R and V179D (n = 41). Site-directed mutagenesis confirmed the importance of these mutations.

| S-EPMC1346823 | biostudies-literature

Nonpolymorphic human immunodeficiency virus type 1 protease and reverse transcriptase treatment-selected mutations.

Project description:The spectrum of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT) mutations selected by antiretroviral (ARV) drugs requires ongoing reassessment as ARV treatment patterns evolve and increasing numbers of protease and RT sequences of different viral subtypes are published. Accordingly, we compared the prevalences of protease and RT mutations in HIV-1 group M sequences from individuals with and without a history of previous treatment with protease inhibitors (PIs) or RT inhibitors (RTIs). Mutations in protease sequences from 26,888 individuals and in RT sequences from 25,695 individuals were classified according to whether they were nonpolymorphic in untreated individuals and whether their prevalence increased fivefold with ARV therapy. This analysis showed that 88 PI-selected and 122 RTI-selected nonpolymorphic mutations had a prevalence that was fivefold higher in individuals receiving ARVs than in ARV-naïve individuals. This was an increase of 47% and 77%, respectively, compared with the 60 PI- and 69 RTI-selected mutations identified in a similar analysis that we published in 2005 using subtype B sequences obtained from one-fourth as many individuals. In conclusion, many nonpolymorphic mutations in protease and RT are under ARV selection pressure. The spectrum of treatment-selected mutations is changing as data for more individuals are collected, treatment exposures change, and the number of available sequences from non-subtype B viruses increases.

| S-EPMC2772298 | biostudies-literature

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