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1.
J Exp Psychol Learn Mem Cogn ; 49(10): 1615-1634, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37307326

RESUMO

Young children can generalize from known to novel, but the underlying mechanism is still debated. Some argue that from an early age generalization is category-based and undergoes little development, while others believe that early generalization is similarity-based, and the use of categories emerges over time. The current research brings new evidence to the debate. In Experiment 1 (N = 118), we presented 3- to 5-year-olds and adults with a category learning task followed by an exemplar generation task. Then, in Experiment 2 (N = 126), we presented the same tasks but provided participants with additional conceptual information about the category members. Our results indicate that early reasoning undergoes dramatic development: whereas young children rely mostly on salient features, adults rely on category information. These results challenge category-based accounts of early generalization while supporting similarity-based accounts. (PsycInfo Database Record (c) 2023 APA, all rights reserved).


Assuntos
Generalização Psicológica , Aprendizagem , Adulto , Criança , Humanos , Pré-Escolar , Resolução de Problemas , Bases de Dados Factuais
2.
Viruses ; 15(4)2023 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-37112961

RESUMO

Several direct-acting antivirals (DAAs) are available, providing interferon-free strategies for a hepatitis C cure. In contrast to DAAs, host-targeting agents (HTAs) interfere with host cellular factors that are essential in the viral replication cycle; as host genes, they are less likely to rapidly mutate under drug pressure, thus potentially exhibiting a high barrier to resistance, in addition to distinct mechanisms of action. We compared the effects of cyclosporin A (CsA), a HTA that targets cyclophilin A (CypA), to DAAs, including inhibitors of nonstructural protein 5A (NS5A), NS3/4A, and NS5B, in Huh7.5.1 cells. Our data show that CsA suppressed HCV infection as rapidly as the fastest-acting DAAs. CsA and inhibitors of NS5A and NS3/4A, but not of NS5B, suppressed the production and release of infectious HCV particles. Intriguingly, while CsA rapidly suppressed infectious extracellular virus levels, it had no significant effect on the intracellular infectious virus, suggesting that, unlike the DAAs tested here, it may block a post-assembly step in the viral replication cycle. Hence, our findings shed light on the biological processes involved in HCV replication and the role of CypA.


Assuntos
Hepatite C Crônica , Hepatite C , Humanos , Hepacivirus/genética , Antivirais/uso terapêutico , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Hepatite C Crônica/tratamento farmacológico , Proteínas não Estruturais Virais/genética , Hepatite C/tratamento farmacológico
3.
Viruses ; 11(11)2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31717338

RESUMO

RNA viruses are highly successful pathogens and are the causative agents for many important diseases. To fully understand the replication of these viruses it is necessary to address the roles of both positive-strand RNA ((+)RNA) and negative-strand RNA ((-)RNA), and their interplay with viral and host proteins. Here we used branched DNA (bDNA) fluorescence in situ hybridization (FISH) to stain both the abundant (+)RNA and the far less abundant (-)RNA in both hepatitis C virus (HCV)- and Zika virus-infected cells, and combined these analyses with visualization of viral proteins through confocal imaging. We were able to phenotypically examine HCV-infected cells in the presence of uninfected cells and revealed the effect of direct-acting antivirals on HCV (+)RNA, (-)RNA, and protein, within hours of commencing treatment. Herein, we demonstrate that bDNA FISH is a powerful tool for the study of RNA viruses that can provide insights into drug efficacy and mechanism of action.


Assuntos
Antivirais/farmacologia , Hepacivirus , RNA Viral , Linhagem Celular , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Hibridização in Situ Fluorescente/métodos , RNA Viral/efeitos dos fármacos , RNA Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Zika virus/genética , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/virologia
4.
J Biol Chem ; 294(39): 14257-14266, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31383738

RESUMO

Autophagy is a conserved cellular process involving intracellular membrane trafficking and degradation. Pathogens, including hepatitis C virus (HCV), often exploit this process to promote their own survival. The aim of this study was to determine the mechanism by which HCV increases steady-state autophagosome numbers while simultaneously inhibiting flux through the autophagic pathway. Using the lysosomal inhibitor bafilomycin A1, we showed that HCV-induced alterations in autophagy result from a blockage of autophagosome degradation rather than an increase in autophagosome generation. In HCV-infected cells, lysosome function was normal, but a tandem RFP-GFP-LC3 failed to reach the lysosome even under conditions that activate autophagy. Autophagosomes and lysosomes isolated from HCV-infected cells were able to fuse with each other normally in vitro, suggesting that the cellular fusion defect resulted from trafficking rather than an inability of vesicles to fuse. Arl8b is an Arf-like GTPase that specifically localizes to lysosomes and plays a role in autophagic flux through its effect on lysosomal positioning. At basal levels, Arl8b was primarily found in a perinuclear localization and co-localized with LC3-positive autophagosomes. HCV infection increased the level of Arl8b 3-fold and redistributed Arl8b to a more diffuse, peripheral pattern that failed to co-localize with LC3. Knockdown of Arl8b in HCV-infected cells restored autophagosome-lysosome fusion and autophagic flux to levels seen in control cells. Thus, HCV suppresses autophagic flux and increases the steady-state levels of autophagosomes by increasing the expression of Arl8b, which repositions lysosomes and prevents their fusion with autophagosomes.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Autofagossomos/metabolismo , Hepatite C/metabolismo , Lisossomos/metabolismo , Fatores de Ribosilação do ADP/genética , Linhagem Celular Tumoral , Humanos , Transporte Proteico
5.
Antimicrob Agents Chemother ; 59(6): 3482-92, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25845863

RESUMO

While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , RNA Viral/genética , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Carbamatos , Hepacivirus/genética , Imidazóis/farmacologia , Pirrolidinas , Ribavirina/farmacologia , Sofosbuvir/farmacologia , Valina/análogos & derivados
6.
Am J Pathol ; 183(6): 1803-1814, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24225087

RESUMO

Hepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3(-/-) mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2(+/-)) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol-induced liver injury.


Assuntos
Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Fatores de Transcrição Forkhead , Hepacivirus/metabolismo , Hepatite C , Hepatopatias Alcoólicas , Animais , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hepacivirus/genética , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/patologia , Humanos , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Camundongos , Camundongos Knockout , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética
7.
Virology ; 444(1-2): 329-36, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23876458

RESUMO

BACKGROUND: We investigated the frequency of RAVs among patients failing to achieve SVR in two clinical trials. We also investigated the impact of interferon responsiveness on RAVs and specific baseline RAVs relationship with boceprevir treatment failure. METHODS: Data are from 1020 patients enrolled into either SPRINT-2 or RESPOND-2; patients received a 4-week PR lead-in prior to receiving boceprevir or placebo. RAVs were analyzed via population-based sequence analysis of the NS3 protease gene (success rate of >90% at a virus level of ≥ 10,000IU/mL) RESULTS: The high SVR rate in patients who received boceprevir resulted in a low rate of RAVs; 7% was detected at baseline in all patients, which rose to 15% after treatment. However, RAVs were detected in 53% of patients that failed to achieve SVR, which declined to 22.8% 6-14 months following cessation of boceprevir therapy. Baseline RAVs alone were not predictive of virologic outcome; poor interferon responsiveness was highly predictive of non-SVR. RAVs were more frequently detected in poor interferon responders. CONCLUSIONS: We detected no association between the presence of baseline amino acid variants at boceprevir resistance-associated loci and outcome in the context of good IFN response.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Hepacivirus/genética , Hepatite C Crônica/virologia , Mutação de Sentido Incorreto , Prolina/análogos & derivados , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos , Antivirais/uso terapêutico , Ensaios Clínicos como Assunto , Hepacivirus/efeitos dos fármacos , Hepacivirus/isolamento & purificação , Hepatite C Crônica/tratamento farmacológico , Humanos , Prolina/farmacologia , Prolina/uso terapêutico , Falha de Tratamento
8.
Antimicrob Agents Chemother ; 57(7): 3250-61, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23629699

RESUMO

While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.


Assuntos
Antivirais/metabolismo , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Indóis/farmacologia , Sulfonamidas/farmacologia , Proteínas não Estruturais Virais/metabolismo , Substituição de Aminoácidos , Animais , Antivirais/farmacocinética , Linhagem Celular Tumoral , Farmacorresistência Viral/genética , Sinergismo Farmacológico , Humanos , Indóis/metabolismo , Indóis/farmacocinética , Interferon-alfa/farmacologia , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Ratos , Ratos Sprague-Dawley , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos
9.
Antivir Ther ; 18(3): 387-97, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23406826

RESUMO

BACKGROUND: Resistance to direct-acting antivirals represents a new challenge in the treatment of chronic hepatitis C. METHODS: SPRINT-1 was a randomized study of treatment-naive patients with genotype (G) 1 hepatitis C infection (n=595) that evaluated the safety and efficacy of boceprevir (BOC) when added to pegylated interferon-α2b plus ribavirin (PR). Plasma samples collected at protocol-specified visits were analysed by population sequencing for detection of BOC-associated resistance-associated variants (RAVs). RESULTS: A total of 17/24 (71%) patients randomized to BOC with baseline RAVs achieved sustained virological response (SVR). V55A/I (n=14), Q41H (n=11) and T54S (n=9) were the most frequently detected polymorphisms at baseline. Seven non-SVR patients with baseline RAVs had V55A (relapse, n=3; breakthrough, n=1; and non-response, n=1) and/or R155K (non-response, n=2). In total, 63/144 (44%) patients with sequenced post-baseline samples (2 SVR, 61 non-SVR) had detectable RAVs after BOC treatment (G1a: R155K [39/49; 80%], V36M [37/49; 76%] and T54S [24/49; 49%]; G1b: T54S [3/11; 27%], T54A [4/11; 35%], A156S [2/11; 18%] and V170A [2/11; 18%]). RAV frequency varied according to the virological response: 90%, 67%, 27% and 37% of breakthrough, incomplete virological response, relapse and non-responder patients, respectively, had post-baseline RAVs present. Similar RAVs were identified in both the PR lead-in and no-lead-in arms and the frequency of post-baseline RAVs was highest in the low-dose ribavirin arm. CONCLUSIONS: SVR rates were not compromised among patients with RAVs at baseline; however, a lower starting mg/kg dose of ribavirin was associated with a higher frequency of post-baseline RAVs.


Assuntos
Substituição de Aminoácidos , Farmacorresistência Viral/genética , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Prolina/análogos & derivados , Ribavirina/uso terapêutico , Quimioterapia Combinada , Genótipo , Humanos , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Polietilenoglicóis/administração & dosagem , Prolina/administração & dosagem , Prolina/uso terapêutico , RNA Viral , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Recidiva , Ribavirina/administração & dosagem , Resultado do Tratamento
10.
Semin Liver Dis ; 31(4): 410-9, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22189980

RESUMO

New treatments for chronic hepatitis C combining direct-acting antivirals (DAAs) with pegylated interferon and ribavirin (PEG-IFN/RBV) have dramatically increased the number of patients whose viral load declines to undetectable levels early in treatment. Most go on to achieve a sustained virologic response, but some patients who maintain undetectable levels of virus throughout treatment later relapse during follow-up. These data suggest that hepatitis C virus (HCV) genomes may persist in form(s) that are refractory to eradication by DAAs and PEG-IFN/RBV. Here we examine the molecular biology of HCV replication and review the clinical virology of relapse for clues as to how the virus might survive months of antiviral therapy to later reappear when treatment is withdrawn.


Assuntos
Antivirais/uso terapêutico , Hepacivirus/patogenicidade , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/fisiopatologia , Adjuvantes Imunológicos/uso terapêutico , Quimioterapia Combinada , Hepacivirus/genética , Humanos , Inibidores de Proteases/uso terapêutico , Recidiva , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
11.
J Biomol Screen ; 16(6): 668-75, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21474836

RESUMO

Infection of certain cell types by HIV results in formation of syncytia. This process can be blocked by antibodies or compounds that prevent interaction of viral envelope protein with host cell receptors. Here the authors describe an automated imaging-based assay for inhibitors of cell-cell fusion mediated by interaction of HIV gp120 with CXCR4 coreceptor. The assay quantifies syncytia formation between U87MG astrocytoma cells constitutively expressing CD4/CXCR4 and morphologically distinct Jurkat T lymphoma cells inducibly expressing HIV env. Each cell type was differentially labeled with vital dyes. Fusion was quantified by measuring size, shape, and color of Jurkat cells and Jurkat-harboring cell syncytia. Dose-response experiments with reference inhibitors AMD 3100 and KRH-1636 yielded potencies consistent with those obtained using standard antiviral assays. This assay complements virus-based infectivity assays for identification of inhibitors of membrane fusion events triggered by interaction of HIV gp120 with host CXCR4.


Assuntos
Células Gigantes/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Processamento de Imagem Assistida por Computador , Receptores CXCR4/metabolismo , Animais , Linhagem Celular , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Células Jurkat , Bibliotecas de Moléculas Pequenas/farmacologia
13.
Virology ; 400(1): 145-55, 2010 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-20172579

RESUMO

The HIV-1 CCR5 co-receptor is a member of the chemokine receptor family of G-protein coupled receptors; for which a number of small molecule antagonists, such as vicriviroc (VCV), have been developed to inhibit HIV-1 R5-tropic replication. In this study, we analyzed an HIV-1 subtype D envelope gene from a clinical trial subject who developed complete resistance to VCV. The HIV-1 resistant envelope has six predominant amino acid changes in the V3 loop, together with one change in the C4 domain of gp120, which are fully responsible for the resistance phenotype. V3 loop mutations Q315E and R321G are essential for resistance to VCV, whereas E328K and G429R in C4 contribute significantly to the infectivity of the resistant variant. Collectively, these amino acid changes influenced the interaction of gp120 with both the N-terminus and ECL2 region of CCR5.


Assuntos
Antagonistas dos Receptores CCR5 , Proteína gp120 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação , Fragmentos de Peptídeos/genética , Piperazinas/farmacologia , Pirimidinas/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Anticorpos Monoclonais , Linhagem Celular , Farmacorresistência Viral/genética , Farmacorresistência Viral/fisiologia , Anticorpos Anti-HIV , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/fisiologia , HIV-1/patogenicidade , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Receptores CCR5/química , Internalização do Vírus
14.
ACS Med Chem Lett ; 1(2): 64-9, 2010 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24900178

RESUMO

Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (∼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.

15.
Virology ; 395(2): 268-79, 2009 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-19846188

RESUMO

Previous studies have demonstrated that HIV-1 develops resistance to CCR5 antagonists by gaining the ability to use drug-occupied co-receptor. However, the effects of CCR5 antagonists on the affinity of virus-co-receptor interactions have been difficult to quantify. We developed a pharmacological model for allosteric interaction at G-protein coupled receptors to analyze the effect of different CCR5 antagonists on infection by three laboratory adapted viruses with low, moderate and high susceptibility to the inhibitors. Infection data for these viruses fitted a model in which susceptibility to inhibition by CCR5 antagonists was directly related to fold reduction in virus affinity for CCR5. Dissociation constants for CCR5 antagonists calculated from the modeled data were consistent with values obtained by standard methods, suggesting that this approach can quantify pharmacologically relevant changes in co-receptor:ligand affinity in the context of infection of whole cells by authentic HIV-1 particles.


Assuntos
Fármacos Anti-HIV/farmacologia , Antagonistas dos Receptores CCR5 , Farmacorresistência Viral , HIV-1/efeitos dos fármacos , Regulação Alostérica , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Receptores Acoplados a Proteínas G/metabolismo
16.
J Virol ; 83(23): 12151-63, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19776131

RESUMO

Vicriviroc (VCV) is a small-molecule CCR5 coreceptor antagonist currently in clinical trials for treatment of R5-tropic human immunodeficiency virus type 1 (HIV-1) infection. With this drug in development, identification of resistance mechanisms to VCV is needed to allow optimal outcomes in clinical practice. In this study we further characterized VCV resistance in a lab-adapted, VCV-resistant RU570 virus (RU570-VCV(res)). We show that K305R, R315Q, and K319T amino acid changes in the V3 loop, along with P437S in C4, completely reproduced the resistance phenotype in a chimeric ADA envelope containing the C2-V5 region from RU570 passage control gp120. The K305R amino acid change primarily impacted the degree of resistance, whereas K319T contributed to both resistance and virus infectivity. The P437S mutation in C4 had more influence on the relative degree of virus infectivity, while the R315Q mutation contributed to the virus concentration-dependent phenotypic resistance pattern observed for RU570-VCV(res). RU570-VCV(res) pseudovirus entry with VCV-bound CCR5 was dramatically reduced by Y10A, D11A, Y14A, and Y15A mutations in the N terminus of CCR5, whereas these mutations had less impact on entry in the absence of VCV. Notably, an additional Q315E/I317F substitution in the crown region of the V3 loop enhanced resistance to VCV, resulting in a stronger dependence on the N terminus for viral entry. By fitting the envelope mutations to a molecular model of a recently described docked N-terminal CCR5 peptide consisting of residues 2 to 15 in complex with HIV-1 gp120 CD4, potential new interactions in gp120 with the N terminus of CCR5 were uncovered. The cumulative results of this study suggest that as the RU570 VCV-resistant virus adapted to use the drug-bound receptor, it also developed an increased reliance on the N terminus of CCR5.


Assuntos
Substituição de Aminoácidos/genética , Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/genética , HIV-1/efeitos dos fármacos , Mutação de Sentido Incorreto , Piperazinas/farmacologia , Pirimidinas/farmacologia , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Receptores CCR5/metabolismo , Receptores de HIV/metabolismo , Internalização do Vírus
17.
Hepatology ; 50(6): 1709-18, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19787809

RESUMO

UNLABELLED: Boceprevir is a hepatitis C virus (HCV) nonstructural protein (NS) 3/4A protease inhibitor that is currently being evaluated in combination with peginterferon alfa-2b and ribavirin in phase 3 studies. The clinical resistance profile of boceprevir is not characterized in detail so far. The NS3 protease domain of viral RNA was cloned from HCV genotype 1-infected patients (n = 22). A mean number of 47 clones were sequenced before, at the end, and after treatment with 400 mg boceprevir twice or three times daily for 14 days for genotypic, phenotypic, and viral fitness analysis. At the end of treatment, a wild-type an NS3 protease sequence was observed with a mean frequency of 85.9%. In the remaining isolates, five previously observed resistance mutations (V36M/A, T54A/S, R155K/T, A156S, V170A) and one mutation (V55A) with unknown resistance to boceprevir were detected either alone or in combination. Phenotypic analysis in the HCV replicon assay showed low (V36G, T54S, R155L; 3.8- to 5.5-fold 50% inhibitory concentration [IC(50)]), medium (V55A, R155K, V170A, T54A, A156S; 6.8- to 17.7-fold IC(50)) and high level (A156T; >120-fold IC(50)) resistance to boceprevir. The overall frequency of resistant mutations and the level of resistance increased with greater declines in mean maximum HCV RNA levels. Two weeks after the end of treatment, the frequency of resistant variants declined and the number of wild-type isolates increased to 95.5%. With the exception of V36 and V170 variants all resistant mutations declined by more than 50%. Mathematical modeling revealed impaired replicative fitness for all single mutations, whereas for combined mutations a relative increase of replication efficiency was suggested. CONCLUSION: During boceprevir monotherapy, resistance mutations at six positions within the NS3 protease were detected by way of clonal sequence analysis. All mutations are associated with reduced replicative fitness estimated by mathematical modeling and show cross-resistance to telaprevir.


Assuntos
Antivirais/uso terapêutico , Hepatite C/tratamento farmacológico , Prolina/análogos & derivados , Inibidores de Proteases/uso terapêutico , Proteínas não Estruturais Virais/antagonistas & inibidores , Método Duplo-Cego , Farmacorresistência Viral , Genótipo , Hepatite C/virologia , Humanos , Modelos Teóricos , Mutação , Oligopeptídeos/uso terapêutico , Fenótipo , Prolina/uso terapêutico , Proteínas não Estruturais Virais/genética
18.
Nucleic Acids Res ; 37(10): e74, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19395595

RESUMO

A major challenge to successful antiviral therapy is the emergence of drug-resistant viruses. Recent studies have developed several automated analyses of HIV sequence polymorphism based on calculations of selection pressure (K(a)/K(s)) to predict drug resistance mutations. Similar resistance analysis programs for HCV inhibitors are not currently available. Taking advantage of the recently available sequence data of patient HCV samples from a Phase II clinical study of protease inhibitor boceprevir, we calculated the selection pressure for all codons in the HCV protease region (amino acid 1-181) to identify potential resistance mutations. The correlation between mutations was also calculated to evaluate linkage between any two mutations. Using this approach, we identified previously known major resistant mutations, including a recently reported mutation V55A. In addition, a novel mutation V158I was identified, and we further confirmed its resistance to boceprevir in protease enzyme and replicon assay. We also extended the approach to analyze potential interactions between individual mutations and identified three pairs of correlated changes. Our data suggests that selection pressure-based analysis and correlation mapping could provide useful tools to analyze large amount of sequencing data from clinical samples and to identify new drug resistance mutations as well as their linkage and correlations.


Assuntos
Análise Mutacional de DNA/métodos , Farmacorresistência Viral/genética , Mutação , Serina Endopeptidases/genética , Proteínas não Estruturais Virais/genética , Antivirais/química , Antivirais/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Prolina/análogos & derivados , Prolina/química , Prolina/farmacologia , Serina Endopeptidases/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química
19.
Antimicrob Agents Chemother ; 53(2): 401-11, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18936191

RESUMO

HCV-796 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase, and boceprevir is an inhibitor of the NS3 serine protease. The emergence of replicon variants resistant to the combination of HCV-796 and boceprevir was evaluated. Combining the inhibitors greatly reduced the frequency with which resistant colonies arose; however, some resistant replicon cells could be isolated by the use of low inhibitor concentrations. These replicons were approximately 1,000-fold less susceptible to HCV-796 and 9-fold less susceptible to boceprevir. They also exhibited resistance to anthranilate nonnucleoside inhibitors of NS5B but were fully sensitive to inhibitors of different mechanisms: a pyranoindole, Hsp90 inhibitors, an NS5B nucleoside inhibitor, and pegylated interferon (Peg-IFN). The replicon was cleared from the combination-resistant cells by extended treatment with Peg-IFN. Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons. These changes could be selected together and coexist in the same genome. The replicon bearing both changes exhibited reduced sensitivity to inhibition by HCV-796 and boceprevir but had a reduced replicative capacity.


Assuntos
Antivirais/farmacologia , Benzofuranos/farmacologia , Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Inibidores da Síntese de Ácido Nucleico , Prolina/análogos & derivados , Inibidores de Proteases/farmacologia , Replicon/genética , Sulfonamidas/farmacologia , Linhagem Celular , Clonagem Molecular , Eletroporação , Variação Genética , Humanos , Interferons/farmacologia , Mutagênese/efeitos dos fármacos , Mutação/genética , Prolina/farmacologia , RNA Viral/biossíntese , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
J Virol ; 82(13): 6492-500, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18448546

RESUMO

Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.


Assuntos
Infecções por Adenovirus Humanos/fisiopatologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Infecções por Adenovirus Humanos/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Transporte Biológico Ativo/fisiologia , Proteínas do Capsídeo/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , beta Carioferinas/isolamento & purificação , beta Carioferinas/metabolismo
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