RESUMEN
Thermal shift assay (TSA) is a versatile biophysical technique for studying protein interactions. Here, we report a free, open-source software tool TSAR (Thermal Shift Analysis in R) to expedite and automate the analysis of thermal shift data derived either from individual experiments or large screens of chemical libraries. The TSAR package incorporates multiple, dynamic workflows to facilitate the analysis of TSA data and returns publication-ready graphics or processed results. Further, the package includes a graphic user interface (GUI) that enables easy use by non-programmers, aiming to simplify TSA analysis while diversifying visualization. To exemplify the utility of TSAR we screened a chemical library of vitamins to identify molecules that interact with the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1). Our data show that hexameric CA interacts with folic acid in vitro.
RESUMEN
The nonstructural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARSCoV) is a helicase that separates doublestranded RNA or DNA with a 5'3' polarity, using the energy of nucleotide hydrolysis. We have previously determined the minimal mechanism of helicase function by nsp13 where we demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 basepairs each with a catalytic rate of 30 steps per second. In that study we used different constructs of nsp13 (GST and H6 constructs). GSTnsp13 showed much more efficient nucleic acid unwinding than the H6tagged counterpart. At 0.1 second, more than 50% of the ATP is hydrolyzed by GSTnsp13 compared to less than 5% ATP hydrolysis by H6nsp13. Interestingly, the two constructs have the same binding affinity for nucleic acids. We, therefore propose that the difference in the catalytic efficiency of these two constructs is due to the interference of ATP binding by the histidine tag at the aminoterminus of nsp13.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Adenosina Trifosfato/metabolismo , ADN Helicasas/genética , Ácidos Nucleicos/metabolismo , Unión ProteicaRESUMEN
KD-247 is a humanized monoclonal antibody that targets the third hypervariable (V3) loop of gp120. It can efficiently neutralize a broad panel of clade B, but not non-clade B, HIV-1 isolates. To overcome this limitation, we are seeking to prepare genetically-engineered single-chain variable fragments (scFvs) of KD-247 that will have broader neutralizing activity against both clade B and non-clade B HIV-1 isolates. Initial attempts of optimizing the expression of KD-247 scFv have resulted in the formation of insoluble protein. Therefore, we have established purification protocols to recover, purify, and refold the KD-247 scFv from inclusion bodies. The protocol involved step-wise refolding of denatured scFv by dilution, dialysis, and on-column nickel-affinity purification. Monomeric scFv was further purified by size-exclusion chromatography. Using far UV circular dichroism (CD) spectroscopy we confirmed the expected beta-sheet profile of the refolded KD-247 scFv. Importantly, the refolded KD-247 scFv showed neutralizing activity against replication-competent HIV-1 BaL and JR-FL Env pseudotyped HIV-1, at potency comparable to that of the native full-size KD-247 antibody. Ongoing studies focus on the application of this system in generating KD-247 scFv variants with the ability to neutralize clade B and non-clade B HIV-1 isolates.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Anti-VIH/metabolismo , Humanos , Pliegue de Proteína , Anticuerpos de Cadena Única/metabolismoRESUMEN
4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this study we tested EFdA-triphosphate (TP) together with a related compound, ENdA-TP (4'-ethynyl-2-amino-2'-deoxdyadenosine triphosphate) against HIV-1 RTs that carry clinically relevant drug resistance mutations: N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V5I/F77L/F116Y/Q151M. We demonstrate that these enzymes remain susceptible to TDRTIs. Similar to WT RT, the N348I RT is inhibited by EFdA mainly at the point of incorporation through decreased translocation. In addition, the N348I substitution decreases the RNase H cleavage of DNA terminated with EFdA-MP (T/P(EFdA-MP)). Moreover, N348I RT unblocks EFdA-terminated primers with similar efficiency as the WT enzyme, and further enhances EFdA unblocking in the background of AZT-resistance mutations. This study provides biochemical insights into the mechanism of inhibition of N348I RT by TDRTIs and highlights the excellent efficacy of this class of inhibitors against WT and drug-resistant HIV-1 RTs.
Asunto(s)
Antivirales/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Farmacorresistencia Viral/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , MutaciónRESUMEN
4' Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent inhibitor of HIV reverse transcriptase (RT). We have recently named EFdA a Translocation Defective RT Inhibitor (TDRTI) because after its incorporation in the nucleic acid it blocks DNA polymerization, primarily by preventing translocation of RT on the template/primer that has EFdA at the 3'-primer end (T/PEFdA). The sugar ring conformation of EFdA may also influence RT inhibition by a) affecting the binding of EFdA triphosphate (EFdATP) at the RT active site and/or b) by preventing proper positioning of the 3'-OH of EFdA in T/PEFdA that is required for efficient DNA synthesis. Specifically, the North (C2'-exo/C3'-endo), but not the South (C2'-endo/C3'-exo) nucleotide sugar ring conformation is required for efficient binding at the primer-binding and polymerase active sites of RT. In this study we use nuclear magnetic resonance (NMR) spectroscopy experiments to determine the sugar ring conformation of EFdA. We find that unlike adenosine nucleosides unsubstituted at the 4'-position, the sugar ring of EFdA is primarily in the North conformation. This difference in sugar ring puckering likely contributes to the more efficient incorporation of EFdATP by RT than dATP. In addition, it suggests that the 3'-OH of EFdA in T/PEFdA is not likely to prevent incorporation of additional nucleotides and thus it does not contribute to the mechanism of RT inhibition. This study provides the first insights into how structural attributes of EFdA affect its antiviral potency through interactions with its RT target.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Desoxiadenosinas/química , Desoxiadenosinas/farmacología , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Dominio Catalítico , Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , Humanos , Modelos Moleculares , Conformación Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Retroviral revXerse transcriptases (RTs) have an associated RNase H activity that can cleave RNA-DNA duplexes with considerable precision. We believe that the structure of the RNA-DNA duplexes in the context of RT determines the specificity of RNase H cleavage. To test this idea, we treated three related groups of synthetic RNA-DNA hybrids with either Moloney murine leukemia virus (MLV) RT or human immunodeficiency virus type 1 (HIV-1) RT. All of the hybrids were prepared using the same 81-base RNA template. The first series of RNase H substrates was prepared with complementary DNA oligonucleotides of different lengths, ranging from 6 to 20 nucleotides, all of which shared a common 5' end and were successively shorter at their 3' ends. The second series of oligonucleotides had a common 3' end but shorter 5' ends. The DNA oligonucleotides in the third series were all 20 bases long but had non-complementary stretches at either the 5' end, 3' end, or both ends. Several themes have emerged from the experiments with these RNA-DNA duplexes. (1) Both HIV-1 RT and MLV RT cleave fairly efficiently if the duplex region is at least eight bases long, but not if it is shorter. (2) Although, under the conditions we have used, both enzymes require the substrate to have a region of RNA-DNA duplex, both MLV RT and HIV-1 RT can cleave RNA outside the region that is part of the RNA-DNA duplex. (3) The polymerase domain of HIV-1 RT uses certain mismatched segments of RNA-DNA to position the enzyme for RNase H cleavage, whereas the polymerase domain of MLV RT does not use the same mismatched segments to define the position for RNase H cleavage. (4) For HIV-1 RT, a mismatched region near the RNase H domain can interfere with RNase H cleavage; cleavage is usually (but not always) more efficient if the mismatched segment is deleted. These results are discussed in regard to the structure of HIV-1 RT and the differences between HIV-1 RT and MLV RT.
Asunto(s)
Transcriptasa Inversa del VIH/metabolismo , Virus de la Leucemia Murina de Moloney/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Ribonucleasa H/metabolismo , Secuencia de Aminoácidos , Disparidad de Par Base/genética , Secuencia de Bases , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , ARN/química , ARN/genética , ARN/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por Sustrato , Temperatura , Moldes GenéticosRESUMEN
Treating HIV infections with drugs that block viral replication selects for drug-resistant strains of the virus. Particular inhibitors select characteristic resistance mutations. In the case of the nucleoside analogs 3TC and FTC, resistant viruses are selected with mutations at amino acid residue 184 of reverse transcriptase (RT). The initial change is usually to M184I; this virus is rapidly replaced by a variant carrying the mutation M184V. 3TC and FTC are taken up by cells and converted into 3TCTP and FTCTP. The triphosphate forms of these nucleoside analogs are incorporated into DNA by HIV-1 RT and act as chain terminators. Both of the mutations, M184I and M184V, provide very high levels of resistance in vivo; purified HIV-1 RT carrying M184V and M184I also shows resistance to 3TCTP and FTCTP in in vitro polymerase assays. Amino acid M184 is part of the dNTP binding site of HIV-1 RT. Structural studies suggest that the mechanism of resistance of HIV-1 RTs carrying the M184V or M184I mutation involves steric hindrance, which could either completely block the binding of 3TCTP and FTCTP or allow binding of these nucleoside triphosphate molecules but only in a configuration that would prevent incorporation. The available kinetic data are ambiguous: one group has reported that the primary effect of the mutations is at the level of 3TCTP binding; another, at the level of incorporation. We have approached this problem using assays that monitor the ability of HIV-1 RT to undergo a conformational change upon binding a dNTP. These studies show that both wild-type RT and the drug-resistant variants can bind 3TCTP at the polymerase active site; however, the binding to M184V and M184I is somewhat weaker and is sensitive to salt. We propose that the drug-resistant variants bind 3TCTP in a strained configuration that is salt-sensitive and is not catalytically competent.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , VIH-1/enzimología , Lamivudine/metabolismo , Lamivudine/farmacología , Sustitución de Aminoácidos/genética , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Sitios de Unión , Catálisis , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxicitosina/farmacología , Desoxirribonucleótidos/metabolismo , Farmacorresistencia Microbiana/genética , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Modelos Moleculares , Mutación/genética , Ensayos de Protección de Nucleasas , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , ARN/biosíntesis , ARN/química , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/metabolismo , Sales (Química)/farmacología , Electricidad Estática , Moldes Genéticos , Transcripción GenéticaRESUMEN
The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3' terminus of the primer strand. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the "primer grip" (the beta12-beta13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the template-primer (including the primer grip, template grip, and helices alphaH and alphaI of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement of nucleic acid and protein during polymerization.
Asunto(s)
ADN/química , ADN/metabolismo , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , ADN/genética , Cartilla de ADN/genética , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Electricidad EstáticaRESUMEN
Comparison of the recently solved structure of HIV-1 reverse transcriptase (RT)-DNA-dNTP ternary complex with the previously solved structure of RT-DNA binary complex suggests mechanisms by which the HIV-1 RT becomes resistant to nucleoside-analog inhibitors, drugs currently used in the treatment of AIDS.
Asunto(s)
ADN Viral/química , Nucleótidos de Desoxiadenina/química , Nucleótidos de Desoxiguanina/química , Transcriptasa Inversa del VIH/química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Sitios de Unión , ADN Viral/metabolismo , ADN Viral/fisiología , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Predicción , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Sustancias Macromoleculares , Modelos MolecularesRESUMEN
Viral genotype assessment is important for effective clinical management of HIV-1 infected patients, especially when access and/or adherence to antiretroviral treatment is reduced. In this study, we describe development of a matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based viral genotyping assay, termed restriction fragment mass polymorphism (RFMP). This assay is suitable for sensitive, specific and high-throughput detection of multiple drug-resistant HIV-1 variants. One hundred serum samples from 60 HIV-1-infected patients previously exposed to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were analysed for the presence of drug-resistant viruses using the RFMP and direct sequencing assays. Probit analysis predicted a detection limit of 223.02 copies/mL for the RFMP assay and 1268.11 copies/mL for the direct sequencing assays using HIV-1 RNA Positive Quality Control Series. The concordance rates between the RFMP and direct sequencing assays for the examined codons were 97% (K65R), 97% (T69Ins/D), 97% (L74VI), 97% (K103N), 96% (V106AM), 97% (Q151M), 97% (Y181C), 97% (M184VI) and 94% (T215YF) in the reverse transcriptase coding region, and 100% (D30N), 100% (M46I), 100% (G48V), 100% (I50V), 100% (I54LS), 99% (V82A), 99% (I84V) and 100% (L90M) in the protease coding region. Defined mixtures were consistently and accurately identified by RFMP at 5% relative concentration of mutant to wild-type virus while at 20% or greater by direct sequencing. The RFMP assay based on mass spectrometry proved to be sensitive, accurate and reliable for monitoring the emergence and early detection of HIV-1 genotypic variants that lead to drug resistance.
Asunto(s)
Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/genética , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , Polimorfismo de Longitud del Fragmento de Restricción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto , Anciano , Fármacos Anti-VIH/uso terapéutico , Secuencia de Bases , Femenino , Genotipo , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Carga Viral , Adulto JovenRESUMEN
A procedure for the assay of free fatty acids which has been adapted for the assay of phospholipase A2 is described. This consists of the conversion of long chain fatty acids to fatty acyl-CoA using the Mg2(+)-dependent fatty acyl-CoA synthetase, [alpha-32P]ATP and coenzyme A. In order to ensure the complete conversion of the acid to its CoA ester pyrophosphatase is also added to the incubation mixture. AM32P formed in stoichiometric amounts is separated from the remaining AT32P by polyethyleneimine-cellulose thin-layer chromatography and the fatty acid content is calculated from the specific radioactivity of AT32P. As little as 1 to 3 nmol of fatty acids hydrolyzed from any phospholipid using nanogram amounts of phospholipase A2 can be estimated with reliability. The real advantage of the method is that it combines the sensitivity of a radiochemical procedure without having to use radiolabeled substrates for the assay of phospholipases.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Ácidos Grasos no Esterificados/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Radioisótopos de Fósforo/análisis , Coenzima A Ligasas/metabolismo , Hidrólisis , Fosfolipasas A2 , Fosfolípidos/metabolismo , Pirofosfatasas/metabolismo , Especificidad por SustratoRESUMEN
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV type 1 (HIV-1) reverse transcriptase (RT). Yeast grown in the presence of many of these drugs exhibited dramatically increased association of the p66 and p51 subunits of the HIV-1 RT as reported by a yeast two-hybrid assay. The enhancement required drug binding by RT; introduction of a drug-resistance mutation into the p66 construct negated the enhancement effect. The drugs could also induce heterodimerization of dimerization defective mutants. Coimmunoprecipitation of RT subunits from yeast lysates confirmed the induction of heterodimer formation by the drugs. In vitro-binding studies indicate that NNRTIs can bind tightly to p66 but not p51 and then mediate subsequent heterodimerization. This study demonstrates an unexpected effect of NNRTIs on the assembly of RT subunits.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Sustitución de Aminoácidos , Sitios de Unión , Dimerización , Farmacorresistencia Microbiana/genética , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Cinética , Modelos Moleculares , Mutación , Conformación Proteica , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Relación Estructura-ActividadRESUMEN
The synthesis of retroviral DNA is initiated near the 5' end of the RNA. DNA synthesis is transferred from the 5' end to the 3' end of viral RNA in an RNase H-dependent step. In the case of human immunodeficiency virus type 1 (HIV-1) (and certain other retroviruses that have complex secondary structures at the ends of the viral RNA), there is the possibility that DNA synthesis can lead to a self-priming event that would block viral replication. The extent of RNase H cleavage must be sufficient to allow the strand transfer reaction to occur, but not so extensive that self-priming occurs. We have used a series of model RNA substrates, with and without a 5' cap, to investigate the rules governing RNase H cleavage at the 5' end of the HIV-1 genome. These in vitro RNase H cleavage reactions produce an RNA fragment of the size needed to block self-priming but still allow strand transfer. The cleavages seen in vitro can be understood in light of the structure of HIV-1 reverse transcriptase in a complex with an RNA/DNA substrate.
Asunto(s)
Genoma Viral , VIH-1/genética , Ribonucleasa H/metabolismo , Secuencia de Bases , ADN Viral/genética , Hidrólisis , Regiones Promotoras Genéticas , ARN Viral/genéticaRESUMEN
Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5'-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Inhibidores de la Transcriptasa Inversa/metabolismo , Nucleótidos de Timina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Fármacos Anti-VIH/farmacología , Didesoxinucleótidos , Resistencia a Medicamentos , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , Humanos , Modelos Moleculares , Mutagénesis , Conformación Proteica , Inhibidores de la Transcriptasa Inversa/farmacología , Nucleótidos de Timina/farmacología , Zidovudina/farmacologíaRESUMEN
In order to determine the catalytic role of Arg72 of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the kcat of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in kcat was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg72 participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PPi.Arg72 is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.
Asunto(s)
VIH-1/enzimología , VIH-1/genética , ADN Polimerasa Dirigida por ARN/genética , Arginina/genética , Sitios de Unión , Catálisis , Difosfatos/metabolismo , Transcriptasa Inversa del VIH , Técnicas In Vitro , Cinética , Mutagénesis Sitio-Dirigida , Poli A/metabolismo , Poli C/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Inhibidores de la Transcriptasa Inversa , Especificidad por SustratoRESUMEN
In order to define the role of Gln151 in the polymerase function of HIV-1 RT, we carried out site-directed mutagenesis of this residue by substituting it with a conserved (Q151N) and a nonconserved residue (Q151A). Q151N exhibited properties analogous to those of the wild-type enzyme, while Q151A has severely impaired polymerase activity. The Q151A mutant exhibited a 15-100-fold reduction in kcat with RNA [poly(rC) and poly(rA)] templates, while only a 5-fold reduction could be seen with the DNA [poly(dC)] template. Most interestingly, the affinity of the Q151A mutant for dNTP substrate remained unchanged with RNA templates, but a significant increase in Km was noted with the DNA template. The binding affinity of Q151A for DNA remained unchanged, as judged by photoaffinity cross-linking. However, unlike the wild-type enzyme, the Q151A mutant failed to catalyze the nucleotidyl transferase reaction onto the primer terminus of the covalently immobilized template-primer. The enzyme showed profoundly altered divalent cation preference from Mg2+ to Mn2+. These results strongly implicate Q151 of HIV-1 RT in the substrate dNTP binding function and possibly in the following chemical (catalytic) step. The effects of the mutation seem to be through Q151 of the p66 catalytic subunit, as p66WTt/p51Q151A retains the wild-type kinetic constants and nucleotidyl transferase activity. In contrast, p66Q151A/p51WT is indistinguishable from Q151A (mutated in both subunits). A model of the ternary complex (enzyme-template-primer and dNTP) has been used to infer the possible mode by which Q151 may interact with the base moiety of the substrate as well as with Arg72, a residue present within the active site of HIV-1 RT.
Asunto(s)
Desoxirribonucleótidos/metabolismo , Glutamina/metabolismo , VIH-1/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cationes Bivalentes , Clonación Molecular , Transcriptasa Inversa del VIH , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Nucleotidiltransferasas/metabolismo , ADN Polimerasa Dirigida por ARN/genética , Especificidad por SustratoRESUMEN
We have examined amino acid substitutions at residues 115 and 116 in the reverse transcriptase (RT) of HIV-1. A number of properties were examined, including polymerization and processivity on both DNA and RNA templates, strand displacement, ribonucleotide misincorporation, and resistance to nucleoside analogs. The RT variants Tyr-115-Phe and Phe-116-Tyr are similar to wild-type HIV-1 RT in most, but not all, respects. In contrast, the RT variant Tyr-115-Val is significantly impaired in polymerase activity compared with wild-type RT; however, Tyr-115-Val is able to incorporate ribonucleotides as well as deoxyribonucleotides during polymerization and is resistant to a variety of nucleoside analogs.
Asunto(s)
Desoxirribonucleótidos/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Sitios de Unión , Desoxirribonucleótidos/genética , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Cinética , Inhibidores de la Transcriptasa Inversa/farmacologíaRESUMEN
The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12O36]16- (polyoxometalate I) and [(O3PCH2PO3)4W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 microM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme-polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules.
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ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Óxidos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Compuestos de Tungsteno/farmacología , Sitios de Unión , Humanos , Modelos MolecularesRESUMEN
When human immunodeficiency virus type 1 (HIV-1) is selected for resistance to 3TC, the methionine normally present at position 184 is replaced by valine or isoleucine. Position 184 is the X of the conserved YXDD motif; positions 185 and 186 form part of the triad of aspartic acids at the polymerase active site. Structural and biochemical analysis of 3TC-resistant HIV-1 reverse transcriptase (RT) led to a model in which a beta-branched amino acid at position 184 would act as a steric gate. Normal deoxynucleoside triphosphates (dNTPs) could still be incorporated; the oxathiolane ring of 3TCTP would clash with the beta branch of the amino acid at position 184. This model can also explain 3TC resistance in feline immunodeficiency virus and human hepatitis B virus. However, it has been reported (14) that murine leukemia viruses (MLVs) with valine (the amino acid present in the wild type), isoleucine, alanine, serine, or methionine at the X position of the YXDD motif are all resistant to 3TC. We prepared purified wild-type MLV RT and mutant MLV RTs with methionine, isoleucine, and alanine at the X position. The behavior of these RTs was compared to those of wild-type HIV-1 RT and of HIV-1 RT with alanine at the X position. If alanine is present at the X position, both MLV RT and HIV-1 RT are relatively resistant to 3TCTP in vitro. However, the mutant enzymes were impaired relative to their wild-type counterparts; there appears to be steric hindrance for both 3TCTP and normal dNTPs.
Asunto(s)
Fármacos Anti-VIH/farmacología , Citidina Trifosfato/farmacología , VIH-1/efectos de los fármacos , Lamivudine/farmacología , Virus de la Leucemia Murina de Moloney/efectos de los fármacos , ADN Polimerasa Dirigida por ARN/genética , Alanina , Animales , Citidina Trifosfato/análogos & derivados , Didesoxinucleótidos , Farmacorresistencia Microbiana , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Isoleucina , Lamivudine/análogos & derivados , Metionina , Conformación Molecular , Virus de la Leucemia Murina de Moloney/enzimología , Virus de la Leucemia Murina de Moloney/genética , Mutación Puntual , ADN Polimerasa Dirigida por ARN/química , Replicación Viral/efectos de los fármacosRESUMEN
Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutations on the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitro HBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by determination of inhibition constants (K(i)). Recognition of the natural substrate, dCTP, was assessed by determination of Km values. The results from the in vitro studies were as follows: (i) dCTP substrate binding was largely unaffected by the mutations, with Km changing moderately, only in a range of 0.6 to 2.6-fold; (ii) K(i)s for 3TCTP and FTCTP against Met552Ile/Val mutant HBV polymerases were increased 8- to 30-fold; and (iii) the Leu528Met mutation had a modest effect on direct binding of these beta-L-oxathiolane ring-containing nucleotide analogs. A three-dimensional homology model of the catalytic core of HBV polymerase was constructed via extrapolation from retroviral reverse transcriptase structures. Molecular modeling studies using the HBV polymerase homology model suggested that steric hindrance between the mutant amino acid side chain and lamivudine or emtricitabine could account for the resistance phenotype. Specifically, steric conflict between the Cgamma2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring (common to both beta-L-nucleoside analogs lamivudine and emtricitabine) is proposed to account for the resistance observed upon Met552Ile/Val mutation. The effects of the Leu528Met mutation, which also occurs near the HBV polymerase active site, appeared to be less direct, potentially involving rearrangement of the deoxynucleoside triphosphate-binding pocket residues. These modeling results suggest that nucleotide analogs that are beta-D-enantiomers, that have the sulfur replaced by a smaller atom, or that have modified or acyclic ring systems may retain activity against lamivudine-resistant mutants, consistent with the observed susceptibility of these mutants to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.