RESUMO
Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a major target for currently approved anti-HIV drugs. These drugs are divided into two classes: nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). This study illustrates the synthesis and biochemical evaluation of a novel bifunctional RT inhibitor utilizing d4T (NRTI) and a TMC-derivative (a diarylpyrimidine NNRTI) linked via a poly(ethylene glycol) (PEG) linker. HIV-1 RT successfully incorporates the triphosphate of d4T-4PEG-TMC bifunctional inhibitor in a base-specific manner. Moreover, this inhibitor demonstrates low nanomolar potency that has 4.3-fold and 4300-fold enhancement of polymerization inhibition in vitro relative to the parent TMC-derivative and d4T, respectively. This study serves as a proof-of-concept for the development and optimization of bifunctional RT inhibitors as potent inhibitors of HIV-1 viral replication.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Primers do DNA , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/isolamento & purificação , Desenho de Fármacos , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/isolamento & purificação , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Espectrometria de Massas , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/isolamento & purificação , Polietilenoglicóis/farmacologia , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos , Difração de Raios XRESUMO
Drug resistance testing is an important tool in the management of HIV-1 infection. As access to genotypic resistance assays is limited in low- and middle-income settings, alternatives are warranted. The aim of the study was to adapt a phenotypic drug susceptibility assay, ExaVir Drug, for detection of resistance to the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) etravirine (ETR). Five NNRTI resistant mutant forms of RT were produced (L100I, K103N, L100I/K103N, Y181C, V179D) in order to validate the assay for ETR. Furthermore, HIV-1 RT was purified from plasma samples (n = 28) obtained from treatment naïve and experienced HIV-1 infected patients, and ETR drug susceptibility (IC(50)) was estimated. The direct sequencing of the pol gene was performed. The recombinant RT mutants had the expected changes in drug sensitivity patterns. The RTs isolated from plasma of therapy naïve individuals showed low IC(50) for ETR. In the plasma virus from treatment experienced patients with Y181C, A98G, V108I, and/or K101E mutations in the pol gene, higher IC(50) values were found in line with reduced susceptibility data for ETR. This study demonstrates that ExaVir® Drug, a simple enzymatic phenotypic assay, can be used for detection of ETR resistance, including cross-resistance to other NNRTIs, in clinical samples. A further evaluation is needed to define clinical cut-offs; however the assay is an alternative to more costly HIV drug resistance tests, especially in low-income countries.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Piridazinas/farmacologia , Transcriptase Reversa do HIV/isolamento & purificação , HIV-1/isolamento & purificação , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana/métodos , Nitrilas , Pirimidinas , Análise de Sequência de DNA , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genéticaRESUMO
Lectins/hemagglutinins are a class of sugar-binding proteins which agglutinate cells and/or precipitate glycoconjugates. They occur widely in plants but manifest significant differences in activities, which means only a few of them own exploitable potentials. The objective of this study was to find and characterize a multifunctional plant lectin with high potential values in food chemistry and medicine. A 60-kDa lectin from Phaseolus vulgaris L. cv. Extralong Autumn Purple Bean (EAPL) was purified by liquid chromatography, and the sequence of its first 20 N-terminal amino acids was ANEIYFSFQRFNETNLILQR. It was galactose-specific and manifested hemagglutinating activity toward erythrocytes of rabbit, rat, mouse, and human ABO blood types. EAPL manifested anti-HIV-1-RT activity, and it could inhibit the proliferation of human tumor cells by inducing the production of apoptotic bodies. The nitric oxide-inducing activity of EAPL may find application in tumor therapy.
Assuntos
Fabaceae/química , Transcriptase Reversa do HIV/isolamento & purificação , Lectinas/isolamento & purificação , Phaseolus/química , Sequência de Aminoácidos , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/fisiologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Hemaglutinação/efeitos dos fármacos , Humanos , Lectinas/farmacologia , Camundongos , Dados de Sequência Molecular , Neoplasias Nasofaríngeas , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , RNA Mensageiro/genética , Coelhos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estações do AnoRESUMO
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays an essential role in the life cycle of the virus. Therefore, RT has been a primary target in the development of antiviral agents against HIV-1. Given the prevalence of resistant viruses, evaluation of the resistance profile of potential drug candidates is a key step in drug development. A simplified RT purification protocol would facilitate this process, as it provides an efficient method by which to purify RT variants for compound evaluation. Traditional purification protocols require the use of several columns to purify untagged RT. The entire procedure usually requires at least one week to complete. Herein, we report two novel methods that enable us to purify highly active RT in either one or two steps. First, a one-step purification protocol was developed by employing an affinity column that was prepared by conjugating an RNase H specific inhibitor (RNHI) with NHS-activated resin. Cell lysate containing RT was loaded onto the column followed by washing in the presence of 2mM Mn(2+). The RT retained in the column was eluted after soaking overnight in 10mM EDTA to retrieve the Mn(2+). In the other method, a vector was constructed that encodes RT fused to cleavable intein and AviTag (a biotin tag) sequences at the C-terminus. Cell lysate containing biotinylated RT was passed through a DE-52 column and then loaded onto an avidin column. Untagged RT was released from the column by reductive cleavage of the intein by DTT. These two methods significantly shorten the time required to purify untagged WT and mutant RTs.
Assuntos
Antivirais/farmacologia , Cromatografia de Afinidade/métodos , Transcriptase Reversa do HIV/isolamento & purificação , HIV-1/efeitos dos fármacos , Antivirais/uso terapêutico , Sequência de Bases , Vetores Genéticos/efeitos dos fármacos , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Humanos , Ribonuclease H/genéticaRESUMO
Non-nucleoside reverse transcriptase inhibitor (NNRTI) therapy failed in 30 patients with the typical human immunodeficiency virus type 1 reverse transcriptase K103N mutation, detected using standard genotyping. Following discontinuation of NNRTI therapy for a median of 55.9 weeks and a decrease of K103N mutant species to undetectable levels in plasma RNA, minority K103N species remained detectable, by allele-specific PCR, for longer periods of time and at higher frequency, in peripheral blood mononuclear cell (PBMC) DNA than in plasma RNA (76.7% and 46.7% of samples with residual K103N species detected at median frequencies of 18.0% and 3.8%, respectively). Analysis of PBMC DNA should be considered when searching for residual K103N mutant species in patients previously exposed to NNRTIs.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/genética , Leucócitos Mononucleares/virologia , Inibidores da Transcriptase Reversa/uso terapêutico , Fármacos Anti-HIV/administração & dosagem , DNA Viral/sangue , DNA Viral/genética , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/isolamento & purificação , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Técnicas de Diagnóstico Molecular , Mutação , Reação em Cadeia da Polimerase , RNA Viral/sangue , RNA Viral/genética , Inibidores da Transcriptase Reversa/administração & dosagemRESUMO
Native gel electrophoresis is used as a tool to assess structural differences in proteins. This article presents an application to separate oligomeric forms of proteins such as human immunodeficiency virus type 1 (HIV-1) reverse transcriptase monomers and homodimers. Technical difficulties encountered with various native gel techniques and ways to circumvent them are described.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Transcriptase Reversa do HIV/química , Dimerização , Transcriptase Reversa do HIV/isolamento & purificação , Humanos , Indicadores e Reagentes/química , Corantes de Rosanilina/químicaRESUMO
The side chain of Val75 stabilizes the fingers subdomain of the human immunodeficiency virus type 1 reverse transcriptase (RT), while its peptide backbone interacts with the single-stranded DNA template (at nucleotide +1) and with the peptide backbone of Gln151. Specific DNA polymerase activities of mutant RTs bearing amino acid substitutions at position 75 (i.e., V75A, V75F, V75I, V75L, V75M, V75S and V75T) were relatively high. Primer extension experiments carried out in the absence of one deoxyribonucleoside-triphosphate suggested that mutations did not affect the accuracy of the RT, except for V75A, V75F, V75I, and to a lesser extent V75T. The fidelity of RTs bearing mutations V75F and V75I increased 1.8- and 3-fold, respectively, as measured by the M13 lacZ alpha forward mutation assay, while V75A showed 1.4-fold decreased accuracy. Steady- and pre-steady-state kinetics demonstrated that the increased fidelity of V75I and V75F was related to their decreased ability to extend mismatched template-primers, while misincorporation efficiencies were not significantly affected by mutations. The increased mispair extension fidelity of mutant V75I RT could be attributed to the nucleotide affinity loss, observed in reactions with mismatched template-primers. Altogether, these data suggest that Val75 interactions with the 5' template overhang are important determinants of fidelity.
Assuntos
Substituição de Aminoácidos , DNA Viral/biossíntese , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , Valina/metabolismo , Bacteriófago M13/genética , Pareamento Incorreto de Bases , Sítios de Ligação , Catálise , Cristalografia por Raios X , Primers do DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Transcriptase Reversa do HIV/isolamento & purificação , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Humanos , Cinética , Modelos Químicos , Modelos Moleculares , Mutagênese Insercional , Plasmídeos , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Moldes GenéticosRESUMO
The non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI)-binding pocket of HIV-1 RT spans codons 100-110, 180-190 and 220-240 and mutations in these domains are responsible for HIV-1 NNRTI resistance. Recombinant HIV-1 strains carrying G190S/A/E, G190S+T215Y, T215Y and K103N mutations were constructed to evaluate susceptibility to both NNRTIs and nucleoside RT inhibitors (NRTIs). In addition, purified recombinant RT enzymes were obtained to determine the degree of in vitro inhibition by drugs of both classes. High-level resistance to nevirapine and moderate level resistance to both stavudine and zidovudine were associated with G190S/A/E substitutions. The simultaneous presence of G190S and T215Y decreased stavudine and zidovudine susceptibility more than T215Y alone. On the other hand, G190S was associated with a marked decrease in RT catalytic efficiency, while T215Y showed a more limited effect. Interestingly, the simultaneous presence of G190S and T215Y was associated with a reduction in the impairment of the G190S-mutated enzyme. Mutations in the HIV-1 RT NNRTI binding pocket may be associated with cross-resistance to NRTI. Selection of double mutants, with further decrease in NRTI susceptibility, might be favoured by the compensatory effect of T215Y on the reduction of RT catalytic efficiency associated with G190S.
Assuntos
Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Mutação , Inibidores da Transcriptase Reversa/farmacologia , Estavudina/farmacologia , Zidovudina/farmacologia , Substituição de Aminoácidos , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/isolamento & purificação , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Nevirapina/farmacologiaRESUMO
We have recently demonstrated that HIV-1 RT mutants characterized by low dNTP binding affinity display significantly reduced dNTP incorporation kinetics in comparison to wild-type RT. This defect is particularly emphasized at low dNTP concentrations where WT RT remains capable of efficient synthesis. Kinetic interference in DNA synthesis can induce RT pausing and slow down the synthesis rate. RT stalling and slow synthesis rate can enhance RNA template cleavage by RT-RNase H, facilitating transfer of the primer to a homologous template. We therefore hypothesized that reduced dNTP binding RT mutants can promote template switching during minus strand synthesis more efficiently than WT HIV-1 RT at low dNTP concentrations. To test this hypothesis, we employed two dNTP binding HIV-1 RT mutants, Q151N and V148I. Indeed, as the dNTP concentration was decreased, the template switching frequency progressively increased for both WT and mutant RTs. However, as predicted, the RT mutants promoted more transfers compared with WT RT. The WT and mutant RTs were similar in their intrinsic RNase H activity, supporting that the elevated template switching efficiency of the mutants was not the result of the mutations enhancing RNase H activity. Rather, kinetic interference leading to stalled DNA synthesis likely enhanced transfers. These results suggest that the RT-dNTP substrate interaction mechanistically influences strand transfer and recombination of HIV-1 RT.
Assuntos
DNA/química , Desoxirribonucleotídeos/metabolismo , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Sítios de Ligação/genética , DNA/biossíntese , DNA Viral/biossíntese , Escherichia coli/genética , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/isolamento & purificação , Histidina/química , Humanos , Cinética , Mutação , Plasmídeos , Ligação Proteica/genética , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/metabolismo , Moldes GenéticosRESUMO
During HIV replication, reverse transcriptase (RT), assisted by the nucleocapsid protein (NC), converts the genomic RNA into proviral DNA. This process appears to be the major source of genetic variability, as RT can misincorporate nucleotides during minus and plus strand DNA synthesis. To investigate nucleotide addition or substitution by RT, we set up in vitro models containing HIV-1 RNA, cDNA, NC, and various RTs. We used the wild type RT and azidothymidine- and didanosine-resistant RTs, because they represent the major forms of resistant RTs selected in patients undergoing therapies. Results show that all RTs can add nucleotides in a non-template fashion at the cDNA 3'-end, a reaction stimulated by NC. Nucleotide substitutions were examined using in vitro systems where 3'-mutated cDNAs were extended by RT on an HIV-1 RNA template. With NC, RT extension of the mutated cDNAs was efficient, and surprisingly, mutations were frequently corrected. These results suggest for the first time that RT has excision-repair activity that is triggered by NC. Chaperoning of RT by NC might be explained by the fact that NC stabilizes an RT-DNA binary complex. In conclusion, RT-NC interactions appear to play critical roles in HIV-1 variability.
Assuntos
Reparo do DNA , Replicação do DNA , DNA Viral/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Primers do DNA , DNA Viral/genética , Didanosina/farmacologia , Farmacorresistência Viral , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/isolamento & purificação , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Moldes Genéticos , Replicação Viral , Zidovudina/farmacologiaRESUMO
A more sensitive version of ExaVir Load, a test that utilizes reverse transcriptase (RT) activity from virions in plasma to determine HIV-1 viral load, is described. The virions were immobilized on a gel that was washed, followed by lysis of the virions, elution of purified RT, and finally RT activity determination. The changes made to the original test were: (1) improved washing of the immobilized virions by addition of a non-lytic detergent to the wash buffer, (2) improved virion lysis procedure, including changes in salt, detergent and pH, (3) the use of larger sample volumes in the RT assay, and (4) prolonged RT reaction time. The alterations gave a tenfold increased sensitivity compared to the original version. The correlation between RT load by the current test and RNA PCR was the same as previously (r=0.90). Using colorimetric product detection, the average detection limit in a panel of 262 patient plasma from Stockholm was 0.5 fg RT/ml, corresponding to approximately 170 RNA copies/ml. None of 54 HIV-1 RNA negative samples exhibited RT. The amount of RT load positive samples were 19% for samples containing 50-400 RNA, 71% for samples with 400-1,500, and 100% among samples with >8,000 copies/ml (according to Roche Amplicor). The sensitivity could be increased further using fluorimetric detection. In conclusion, the modifications of the test described result in an important increase in sensitivity. It can now be regarded as a competitive alternative method for HIV viral load determinations.
Assuntos
Infecções por HIV/virologia , Transcriptase Reversa do HIV/sangue , HIV-1/fisiologia , Carga Viral/métodos , Fluorometria , Transcriptase Reversa do HIV/isolamento & purificação , Humanos , RNA Viral/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , África do Sul , Espectrofotometria , Estatística como Assunto , SuéciaRESUMO
A highly efficient cell-free translation system has been combined with suppressor tRNA technology to substitute nor-Tyr and 3-fluoro-Tyr in place of Tyr183 at the DNA polymerase active site of p66 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Supplementing the wild-type HIV-1 p51 RT subunit into this translation system permitted reconstitution of the biologically relevant p66/p51 heterodimer harboring Tyr analogs exclusively on the catalytically competent p66 subunit. Addition of an affinity tag at the p66 C-terminus allowed rapid, one-step purification of reconstituted and selectively mutated heterodimer HIV-1 RT via strep-Tactin-agarose affinity chromatography. The purified enzyme was demonstrated to be free of contaminating nucleases, allowing characterization of the DNA polymerase and ribonuclease H activities associated with HIV-1 RT. Preliminary characterization of HIV-1 RT(nor-Tyr) and HIV-1 RT(m-fluoro-Tyr) is presented. The success of this strategy will facilitate detailed molecular analysis of structurally and catalytically critical amino acids via their replacement with closely related, unnatural analogs.
Assuntos
Substituição de Aminoácidos , Transcriptase Reversa do HIV/isolamento & purificação , Subunidades Proteicas/química , Sistema Livre de Células , Dimerização , Transcriptase Reversa do HIV/química , Humanos , Modelos Biológicos , RNA de Transferência/química , RNA de Transferência/isolamento & purificação , Ribonuclease H/química , Ribonuclease H/isolamento & purificaçãoRESUMO
Emtricitabine [(-)FTC; (-)-beta-L-2'-3'-dideoxy-5-fluoro-3'-thiacytidine] is an oxathiolane nucleoside analog recently approved by the Food and Drug Administration for the treatment of human immunodeficiency virus (HIV). Structurally, (-)FTC closely resembles lamivudine [(-)3TC] except that the former is 5-fluorinated on the cytosine ring. In HIV-1 reverse transcriptase (RT) enzymatic assays, the triphosphate of (-)FTC [(-)FTC-TP] was incorporated into both DNA-DNA and DNA-RNA primer-templates nearly 3- and 10-fold more efficiently than (-)3TC-TP. Animal studies and clinical trial studies have demonstrated a favorable safety profile for (-)FTC. However, a detailed study of the incorporation of (-)FTC-TP by human mitochondrial DNA polymerase gamma, a host enzyme associated with nucleoside toxicity, is required for complete understanding of the molecular mechanisms of inhibition and toxicity. We studied the incorporation of (-)FTC-TP and its enantiomer (+)FTC-TP into a DNA-DNA primer-template by recombinant human mitochondrial DNA polymerase in a pre-steady-state kinetic analysis. (-)FTC-TP was incorporated 2.9 x 10(5)-, 1.1 x 10(5)-, 1.6 x 10(3)-, 7.9 x 10(3)-, and 100-fold less efficiently than dCTP, ddCTP, (+)3TC-TP, (+)FTC-TP, and (-)3TC-TP, respectively. The rate of removal of (-)FTC-MP from the corresponding chain-terminated 24-mer DNA by polymerase gamma's 3'-->5' exonuclease activity was equal to the removal of (+)FTC-MP, 2-fold slower than the removal of (-)3TC-MP and (+)3TC-MP, and 4.6-fold slower than the excision of dCMP. These results demonstrate that there are clear differences between HIV-1 RT and polymerase gamma in terms of preferences for substrate structure.
Assuntos
Antivirais/farmacologia , Antivirais/toxicidade , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos de Desoxicitosina/farmacologia , Nucleotídeos de Desoxicitosina/toxicidade , Transcriptase Reversa do HIV/metabolismo , Mitocôndrias/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/toxicidade , DNA/metabolismo , DNA Polimerase gama , Didesoxinucleotídeos , Transcriptase Reversa do HIV/isolamento & purificação , Humanos , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Retroviruses copy their RNA genome into a DNA molecule, but little is known of the structure of the complex mediating reverse transcription in vivo. We used confocal and electron microscopy to study the structure of human immunodeficiency virus type 1 (HIV-1) intracellular reverse transcription complexes (RTCs). Cytoplasmic extracts were prepared 3, 4, and 16 h after acute infection by Dounce homogenization in hypotonic buffer. RTCs were purified by velocity sedimentation, followed by density fractionation in linear sucrose gradients and dialysis in a large pore cellulose membrane. RTCs had a sedimentation velocity of approximately 350 S and a density of 1.34 g/ml and were active in an endogenous reverse transcription assay. Double labeling of nucleic acids and viral proteins allowed specific visualization of RTCs by confocal microscopy. Electron microscopy revealed that RTCs are large nucleoprotein structures of variable shape consisting of packed filaments ca. 6 nm thick. Integrase and Vpr are associated with discrete regions of the 6-nm filaments. The nucleic acids within the RTC are coated by small proteins distinct from nucleocapsid and are partially protected from nuclease digestion.
Assuntos
Transcriptase Reversa do HIV/ultraestrutura , HIV-1/enzimologia , Nucleoproteínas/ultraestrutura , Linhagem Celular , Centrifugação com Gradiente de Concentração , DNA Viral/química , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/isolamento & purificação , Transcriptase Reversa do HIV/metabolismo , Células HeLa , Humanos , Microscopia Confocal , Microscopia Eletrônica , Conformação de Ácido Nucleico , Nucleoproteínas/química , Nucleoproteínas/isolamento & purificação , Nucleoproteínas/metabolismo , Reação em Cadeia da Polimerase , RNA Viral/química , Transcrição GênicaRESUMO
OBJECTIVE: To demonstrate the use of HIV-1 reverse transcriptase (RT) recovered directly from plasma for phenotypic drug susceptibility testing. METHODS: Plasma from HIV-1 infected individuals with and without drug resistance-associated mutations were selected for the study. The blind coded plasmas were treated to inactivate cellular enzymes. The virions were immobilized on a gel and washed to remove antiretroviral drugs and RT activity blocking antibodies. The immobilized virions were lysed; the viral RT eluted and quantified, all according to the ExaVir Load procedure. The drug sensitivity profiles of each RT were determined using serially diluted drugs and modified Cavidi HS Lenti RT kits. RESULTS: The phenotypic drug sensitivity profiles of the RT and the patterns of drug resistance mutations were highly concordant. Plasma RT from virions devoid of mutations associated with drug resistance had average 50% inhibitory concentrations (IC(50)) of 1.5 +/- 0.93 microM for nevirapine, 0.21 +/- 0.099 microM for efavirenz, 7.1 +/- 3.2 microM for delavirdine, 0.42 +/- 0.15 microM for azidothymidine triphosphate and 0.059 +/- 0.018 microM for didehydrothymidine triphosphate. The increase in IC(50) value for RT with drug resistance associated substitutions was from 3- to more than 65-fold for non-nucleoside inhibitors and between 2- and 30-fold for thymidine analogue drugs. CONCLUSION: RT derived from virions recovered from the plasma of HIV infected individuals can be used for analysis of phenotypic drug susceptibility. The methods presented provide rapid alternatives for analysing phenotypic drug susceptibility especially when the therapy is based on non-nucleoside RT inhibitors and thymidine-analogue drugs.
Assuntos
Transcriptase Reversa do HIV/análise , HIV-1/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Zidovudina/análogos & derivados , Fármacos Anti-HIV/farmacologia , Terapia Antirretroviral de Alta Atividade , Didesoxinucleotídeos , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/isolamento & purificação , Humanos , Reprodutibilidade dos Testes , Estavudina/farmacologia , Nucleotídeos de Timina/farmacologia , Vírion/química , Zidovudina/farmacologiaRESUMO
Template switching during reverse transcription promotes recombination in retroviruses. Efficient switches have been measured in vitro on hairpin-containing RNA templates by a two-step mechanism. Pausing of the reverse transcriptase (RT) at the hairpin base allowed enhanced cleavage of the initial donor RNA template, exposing regions of the cDNA and allowing the acceptor to base pair with the cDNA. This defines the first or docking step. The primer continued synthesis on the donor, transferring or locking in a second step. Here we determine the enzyme-dependent factors that influence template switching by comparing the RTs from human immunodeficiency virus, type 1 (HIV-1), and equine infectious anemia virus (EIAV). HIV-1 RT promoted transfers with higher efficiency than EIAV RT. We found that both RTs paused strongly at the base of the hairpin. While stalled, HIV-1 RT made closely spaced cuts, whereas EIAV RT made only a single cut. Docking occurred efficiently at the multiply cut but not at the singly cut site. HIV-1 nucleocapsid (NC) protein stimulated strand transfers. It improved RNase H activity of both RTs. It allowed the EIAV RT to make a distribution of cuts, greatly stimulating docking at the base of the hairpin. Most likely, it also promoted strand exchange, allowing transfers to be initiated from sites throughout the hairpin. Minor pause sites beyond the base of the hairpin correlated with the locking sites. The strand exchange properties of NC likely promote this step. We present a model that explains the roles of RNase H specificity, template structure, and properties of NC in the two-step transfer reaction.
Assuntos
Transcriptase Reversa do HIV/química , Proteínas do Nucleocapsídeo/química , Recombinação Genética , Retroviridae/genética , Transcriptase Reversa do HIV/isolamento & purificação , Conformação de Ácido Nucleico , RNA Viral/química , Moldes GenéticosRESUMO
Recent crystallographic data suggest that a number of hydrophobic residues seen clustered between the structurally conserved alphabetabetaalpha motif of the palm subdomain and at the junction of palm and fingers subdomains of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) provide an optimal geometry to the alphabeta sandwich of the palm subdomain, which harbors the catalytic site and the primer-binding grip region. This region has also been implicated in binding to the non-nucleoside RT inhibitors. We have evaluated the impact of conserved and nonconserved amino acid substitutions at four hydrophobic positions in this region of HIV-1 RT, in the context of their biochemical characteristics. The residues that have been analyzed include Ile-167, Leu-187, and Val-189 which are located within the alphabetabetaalpha motif, while Trp-153 lies next to the conserved LPQG motif, at the juncture of the palm and fingers subdomains. Our results show that all substitutions at I167 with the exception of I167T were deleterious to enzyme function in contrast to substitutions at V189 which enhanced the enzymatic activity. Ala substitution at residues W153 and L187 also substantially hindered the polymerase function of the enzyme. Further analysis revealed that the defective mutant derivatives of I167 were substantially impaired in their apparent dNTP binding abilities, thereby impacting the geometry of the dNTP binding pocket. The extent of misinsertion and misincorporation was higher in the case of RT variants of W153 and V189, specifically on a DNA template. Interestingly, none of the mutant derivatives of these residues were resistant to nucleoside inhibitors. A salient finding was that all nonconserved mutants of these residues exhibited hypersensitivity to nevirapine. We have analyzed these findings and their significance in the context of the HIV-1 RT structure and propose that these residues exert their effect via their indirect interactions with the template-primer through residues in their vicinity.
Assuntos
Substituição de Aminoácidos , Domínio Catalítico , Sequência Conservada , Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Motivos de Aminoácidos/genética , Substituição de Aminoácidos/genética , Pareamento Incorreto de Bases/genética , Sequência de Bases , Sítios de Ligação/genética , Domínio Catalítico/genética , Sequência Conservada/genética , DNA Viral/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desoxirribonucleosídeos/química , Desoxirribonucleotídeos/química , Didesoxinucleosídeos/química , Vetores Genéticos/síntese química , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/isolamento & purificação , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nevirapina/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Especificidade por Substrato/genética , Moldes GenéticosRESUMO
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates DNA synthesis from the 3' end of human tRNA(Lys3). We have used cis-acting hammerhead ribozymes to produce homogeneous-length transcribed tRNA(Lys3) and have developed conditions for purifying highly structured RNAs on a modified tube-gel apparatus. Titration experiments show that this RNA can assemble into an initiation complex that contains equimolar amounts of HIV-1 RT, transcribed tRNA(Lys3), and chemically synthesized template RNA. We have purified this complex using gel-filtration chromatography and have found that it is homogeneous with respect to molecular weight, demonstrating that the initiation complex forms a single discrete species at micromolar concentrations. When this initiation complex is supplied with deoxynucleotides, essentially all of the tRNA is used as a primer by HIV-1 RT and is fully extended to the 5' end of the template. Thus, in vitro transcribed tRNA can be used efficiently as a primer by HIV-1 RT. We have also obtained crystals of the HIV-1 initiation complex that require the precisely defined ends of this in vitro transcribed tRNA(Lys3) to grow.
Assuntos
Transcriptase Reversa do HIV , RNA de Transferência de Lisina , Sequência de Bases , Cromatografia em Gel , Cristalização , DNA/biossíntese , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/isolamento & purificação , Transcriptase Reversa do HIV/metabolismo , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , RNA Catalítico/metabolismo , RNA de Transferência de Lisina/química , RNA de Transferência de Lisina/isolamento & purificação , RNA de Transferência de Lisina/metabolismo , Moldes Genéticos , Transcrição GênicaRESUMO
Human immunodeficiency virus (HIV)-1 strains have been divided into three groups: main (M), outlier (O), and non-M non-O (N). Biochemical analyses of HIV-1 reverse transcriptase (RT) have been performed predominantly with enzymes derived from HIV-1 group M:subtype B laboratory strains. This study was designed to optimize the expression and to characterize the enzymatic properties of HIV-1 group O RTs as well as chimeric RTs composed of group M and O p66 and p51 subunits. The DNA-dependent DNA polymerase activity on a short heteropolymeric template-primer was similar with all enzymes, i.e. the HIV-1 group O and M and chimeric RTs. Our data revealed that the 51-kDa subunit in the chimeric heterodimer p66(M:B)/p51(O) confers increased heterodimer stability and partial resistance to non-nucleoside RT inhibitors. Chimeric RTs (p66(M:B)/p51(O) and p66(O)/p51(M:B)) were unable to initiate reverse transcription from tRNA(3)(Lys) using HIV-1 group O or group M:subtype B RNA templates. In contrast, HIV-1 group O and M RTs supported (-)-strand DNA synthesis from tRNA(3)(Lys) hybridized to any of their corresponding HIV-1 RNA templates. HIV-2 RT could not initiate reverse transcription on tRNA(3)(Lys)-primed HIV-1 genomic RNA. These findings suggest that the initiation event is conserved between HIV-1 groups, but not HIV types.
Assuntos
Transcriptase Reversa do HIV/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Bases , Primers do DNA , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/isolamento & purificação , HIV-1/genética , HIV-2/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , RNA Viral/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Inibidores da Transcriptase Reversa/farmacologia , Homologia de Sequência do Ácido NucleicoRESUMO
To examine the early events of the life cycle of human immunodeficiency virus type 1 (HIV-1), we analyzed the intracellular complexes mediating reverse transcription isolated from acutely infected cells. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that two species of RTCs are formed but only one species is able to synthesize DNA. Most of the capsid, matrix, and reverse transcriptase (RT) proteins dissociate from the complex soon after cell infection, but Vpr remains associated with the RTC. The RTCs isolated 1, 4, and 7 h after infection are competent for reverse transcription in vitro, indicating that a small proportion of RT remains associated with them. HIV RTCs isolated early after infection have a sedimentation velocity of approximately 560S. Later, different species with a sedimentation velocity ranging from 350S to 100S appear. Nuclear-associated RTCs have a sedimentation velocity of 80S. Shortly after initiation of reverse transcription, the viral strong-stop DNA within the RTC is sensitive to nuclease digestion and becomes protected when reverse transcription is almost completed.