RESUMO
Little is known about respiratory viruses infection in Guinea. Influenza surveillance has not been implemented in Guinea mainly because of the paucity of laboratory infrastructure and capacity. This paper presents the first influenza surveillance data in Guinea.Swabs were obtained from August 2018 through December 2019 at influenza sentinel sites and transported to the Institut National de Santé Publique for testing. Ribonucleic acid was extracted and tested for the presence of influenza A and B by real-time reverse transcription-polymerase chain reaction (RT-PCR). Positive samples were further characterised to determine the subtypes and lineages of influenza viruses.A total of 862 swabs were collected and tested. Twenty-three per cent of samples tested positive for influenza A and B viruses. Characterisation of positive specimens identified influenza A/H1N1pmd09 (2.5%), influenza A/H3N2 (57.3%), influenza B/Victoria lineage (36.7%) and 7 (3.5%) influenza B with undetermined lineage. Influenza B virus activity clustered in August through November while influenza A/H3N2 displayed two clusters of activities that appeared in May through August and November through December.For the first time in Guinea, the epidemiology, diversity and period of circulation of influenza viruses were studied. The results indicate the predominance and the periods of activities of influenza B Victoria lineage and influenza A/H3N2 which are important information for preventive strategies. It is warranted to extend the influenza surveillance to other parts of Guinea to better understand the epidemiology of the viruses and monitor the emergence of influenza strains with pandemic potential.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Monitoramento Epidemiológico , Guiné/epidemiologia , Humanos , Estações do AnoRESUMO
Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma's Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50=1.4 µM), suramin sodium salt (IC50=3.6 µM), NF 023 hydrate (IC50=6.2 µM) and tyrphostin AG 538 (IC50=3.6 µM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Hepacivirus/enzimologia , Ensaios de Triagem em Larga Escala , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaios Enzimáticos , Proteínas de Escherichia coli/metabolismo , Polarização de Fluorescência , RNA Helicases/metabolismo , Bibliotecas de Moléculas Pequenas , Proteínas não Estruturais Virais/metabolismoRESUMO
The antiviral factor CPSF6-358 interferes with the nuclear entry of human immunodeficiency virus type 1 (HIV-1). HIV-1 acquires resistance to CPSF6-358 through the N74D mutation of the capsid (CA), which alters its nuclear entry pathway. Here we show that compared to wild-type (WT) HIV-1, N74D HIV-1 is more sensitive to cyclosporine, has increased sensitivity to nevirapine, and is impaired in macrophage infection prior to reverse transcription. These phenotypes suggest a difference in the N74D reverse transcription complex that manifests early after infection and prior to interaction with the nuclear pore. Overall, our data indicate that N74D HIV-1 replication in transformed cells requires cyclophilin A but is dependent on other interactions in macrophages.
Assuntos
Proteínas do Capsídeo/genética , Ciclofilina A/farmacologia , HIV-1/genética , HIV-1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Mutação , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Animais , Fatores de Restrição Antivirais , Aotidae , Proteínas do Capsídeo/metabolismo , Proteínas de Transporte/farmacologia , Divisão Celular , Linhagem Celular , HIV-1/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
Background: The 2014-2016 West Africa Ebola virus disease outbreak heavily impacted the Republics of Guinea, Sierra Leone, and Liberia. The outbreak uncovered the weaknesses of the public health systems, including inadequately trained and insufficient health personnel as well as limited and poorly equipped health infrastructures. These weaknesses represent significant threats to global health security. In the wake of the outbreak, affected countries made urgent requests for international engagement to help strengthening the public health systems. Methods: This work describes the successful multi-year implementation of a laboratory capacity building program in the Republic of Guinea. The program integrated biorisk and quality management systems training, infectious diseases diagnostic training, facility engineering and maintenance training, and mentorship to strengthen Guinea's bio-surveillance capacity. Results: The major outcome of these efforts was an established and local staff-operated public health laboratory that performs disease surveillance and reporting and diagnostic of priority diseases and pathogens of security concerns. Conclusions: This work has improved the Guinea country's capabilities to address country public health issues and preparedness to respond to future infectious disease threats.
Assuntos
Doença pelo Vírus Ebola , Fortalecimento Institucional , Surtos de Doenças/prevenção & controle , Guiné/epidemiologia , Doença pelo Vírus Ebola/diagnóstico , Humanos , Laboratórios , Libéria , Serra LeoaRESUMO
BACKGROUND: Ebola virus emerged in West Africa in December 2013. The ease of mobility, porous borders, and lack of public health infrastructure led to the largest Ebola virus disease (EVD) outbreak to date. INTERVENTION: The 2013 EVD outbreak signalled the need for laboratory diagnostic capabilities in areas without strong public health systems. As part of the United States' Department of Defense response, MRIGlobal was contracted to design, fabricate, equip, deploy, and operate two mobile diagnostic laboratories (MDLs). The first laboratory analysed blood samples from patients in an adjacent Ebola Treatment Centre (ETC) and buccal swabs from the deceased in the community in Moyamba, Sierra Leone. The second laboratory was deployed to support an ETC in Conakry, Guinea. The Department of Defense provided real-time quantitative reverse transcription polymerase chain reaction assays that were deployed and validated on-site. LESSONS LEARNT: Prompt and accurate molecular diagnostics reduced sample turn-around times from over 24 h to under 4 h. Experienced laboratory staff tested up to 110 samples per day and on-site engineering proved necessary for MDL setup and operation. As the Ebola response slowed, the sustainment of the MDLs' operations was prioritised, including staff training and the transition of the MDLs to local governments. Training programmes for local staff were prepared in Sierra Leone and Guinea. RECOMMENDATIONS: The MRIGlobal MDL team significantly contributed to establishing increased laboratory capacity during the EVD outbreak in West Africa. Using the MDLs for molecular diagnosis is highly recommended until more sustainable solutions can be provided.
RESUMO
Human hepatitis C virus (HCV) does not replicate in mouse hepatocytes. The underlying mechanisms are largely unknown. In this study, we took advantage of a series of direct and unique molecular strategies and dissected the HCV life cycle in human hepatoma Huh7.5 cells and mouse hepatoma Hepa1-6 cells. We showed that HCV entry was restricted in Hepa1-6 cells and was not rescued by expression of human HCV receptors. We also showed that HCV RNA replication was impaired in Hepa1-6 cells. In contrast, we showed that the HCV IRES translation activity and HCV production in Hepa1-6 cells were either comparable to, or even slightly higher than those in Huh7.5 cells. Thus, we conclude that entry and RNA replication are the two major HCV restrictions in mouse cells. These studies provide new insights into HCV interaction with mouse cells and new clues for formulating strategies for development of HCV mouse models.
Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Hepatócitos/virologia , Internalização do Vírus , Replicação Viral , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , RNA Viral/biossínteseRESUMO
Infection with genotype 4 of the Hepatitis C virus is common in Africa and the Mediterranean area, but has also been found at increasing frequencies in injection drug users in Europe and North America. Full length viral sequences to characterize viral diversity and structure have recently become available mostly for subtype 4a, and studies in Egypt and Saudi Arabia, where high proportions of subtype 4a infected patients exist, have begun to establish optimized treatment regimens. However knowledge about other subtype variants of genotype 4 present in less developed African states is lacking. In this study the full coding region from so far poorly characterized variants of HCV genotype 4 was amplified and sequenced using a long range PCR technique. Sequences were analyzed with respect to phylogenetic relationship, possible recombination and prominent sequence characteristics compared to other known HCV strains. We present for the first time two full-length sequences from the HCV genotype 4k, in addition to five strains from HCV genotypes 4d and 4f. Reference sequences for accurate HCV genotyping are required for optimized treatment, and a better knowledge of the global viral sequence diversity is needed to guide vaccines or new drugs effective in the world wide epidemic.
Assuntos
Hepacivirus/classificação , Hepacivirus/genética , Fases de Leitura Aberta/genética , Sequência de Aminoácidos , Sequência de Bases , Genótipo , Hepatite C/virologia , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/métodos , Recombinação Genética , Padrões de Referência , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The Nef protein of human immunodeficiency virus type 1 (HIV-1) has multiple functional domains, is immunogenic, and contains several cytotoxic T lymphocyte (CTL)-targeted epitopes. Several defined subfunctions of Nef are important for the pathogenesis of HIV-1 infection. In this study, we present the genetic diversity of the nef gene of 55 newly derived HIV-1 sequences obtained from Cameroonian patients. Four genetic subtypes and three circulating recombinant forms (CRFs) were identified: subtypes A (11%), G (7.3%), D (5.4%), F1 (1.8%), F2 (5.4%), CRF01_AE (5.4%), CRF02_AG (58.2%), and CRF11_cpx (1.8%). Two isolates clustered distinctly from the known HIV-1 genetic subtypes in nef and were designated as unclassified. Interestingly, the majority of all functional domains including the myristoylation signal, CD4 binding motif, beta turn motif, and the phosphorylation sites were well conserved in our cohort. Putative CTL-epitopic domains of the central portion of Nef were also well conserved, whereas those at the C-term were not. Our study demonstrated that despite high genetic diversity observed in the nef gene, most described functional domains and CTL epitopes were well conserved among Cameroonian HIV-1 subtypes. These findings could be used for the development of antiretroviral-acting therapeutics and anti-HIV-1 vaccines.
Assuntos
Produtos do Gene nef/genética , Adulto , Sequência de Aminoácidos , Camarões , Feminino , Variação Genética , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estudos Prospectivos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 µM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%.
RESUMO
This study examines the specificity and mechanism of action of a recently reported hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase-protease inhibitor (HPI), and the interaction of HPI with the NS3 protease inhibitors telaprevir, boceprevir, danoprevir, and grazoprevir. HPI most effectively reduced cellular levels of subgenomic genotype 4a replicons, followed by genotypes 3a and 1b replicons. HPI had no effect on HCV genotype 2a or dengue virus replicon levels. Resistance evolved more slowly to HPI than telaprevir, and HPI inhibited telaprevir-resistant replicons. Molecular modeling and analysis of the ability of HPI to inhibit peptide hydrolysis catalyzed by a variety of wildtype and mutant NS3 proteins suggested that HPI forms a bridge between the NS3 RNA-binding cleft and an allosteric site previously shown to bind other protease inhibitors. In most combinations, the antiviral effect of HPI was additive with telaprevir and boceprevir, minor synergy was observed with danoprevir, and modest synergy was observed with grazoprevir.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Hepacivirus/química , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Modelos Moleculares , Terapia de Alvo Molecular , Oligopeptídeos/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 µM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 µM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure-activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines.
Assuntos
Antivirais/farmacologia , Azóis/farmacologia , Hepatite C/virologia , Ácidos Nucleicos/metabolismo , Compostos Organosselênicos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Antioxidantes/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Proliferação de Células , Ensaio de Desvio de Mobilidade Eletroforética , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Hepatite C/metabolismo , Humanos , Hidrólise , Isoindóis , Cinética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Modelos Moleculares , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Helicases are ubiquitous motor proteins that separate and/or rearrange nucleic acid duplexes in reactions fueled by adenosine triphosphate (ATP) hydrolysis. Helicases encoded by bacteria, viruses, and human cells are widely studied targets for new antiviral, antibiotic, and anticancer drugs. This review summarizes the biochemistry of frequently targeted helicases. These proteins include viral enzymes from herpes simplex virus, papillomaviruses, polyomaviruses, coronaviruses, the hepatitis C virus, and various flaviviruses. Bacterial targets examined include DnaB-like and RecBCD-like helicases. The human DEAD-box protein DDX3 is the cellular antiviral target discussed, and cellular anticancer drug targets discussed are the human RecQ-like helicases and eIF4A. We also review assays used for helicase inhibitor discovery and the most promising and common helicase inhibitor chemotypes, such as nucleotide analogues, polyphenyls, metal ion chelators, flavones, polycyclic aromatic polymers, coumarins, and various DNA binding pharmacophores. Also discussed are common complications encountered while searching for potent helicase inhibitors and possible solutions for these problems.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , DNA Helicases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Virais/antagonistas & inibidores , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Proteínas de Bactérias/fisiologia , DNA Helicases/fisiologia , Replicação do DNA , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica , Biossíntese de Proteínas , Proteínas Virais/fisiologiaRESUMO
Although antiretroviral therapy (ART) can suppress HIV-1 replication sufficiently to eliminate measurable plasma viremia, infected cells remain and ensure viral recrudescence after discontinuation of ART. We used a macaque model of HIV-1/AIDS to evaluate the location of infected cells during ART. Twelve macaques were infected with RT-SHIVmne, a SIV containing HIV-1 reverse transcriptase, conferring sensitivity to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten to fourteen weeks post-infection, 6 animals were treated with 3 or 4 antiretroviral drugs for 17-20 weeks; 6 control animals remained untreated. Viral DNA (vDNA) and RNA (vRNA) were measured in peripheral blood mononuclear cells (PBMC) and at necropsy in multiple tissues by quantitative PCR and RT-PCR. The majority of virally infected cells were located in lymphoid tissues with variable levels in the gastrointestinal tract of both treated and untreated animals. Tissue viral DNA levels correlated with week 1 plasma viremia, suggesting that tissues that harbor proviral DNA are established within the first week of infection. PBMC vDNA levels did not correlate with plasma viremia or tissue levels of vDNA. vRNA levels were high in lymphoid and gastrointestinal tissues of the untreated animals; animals on ART had little vRNA expressed in tissues and virus could not be cultured from lymph node resting CD4+ cells after 17-20 weeks on ART, indicating little or no ongoing viral replication. Strategies for eradication of HIV-1 will need to target residual virus in ART suppressed individuals, which may not be accurately reflected by frequencies of infected cells in blood.
Assuntos
Antirretrovirais/farmacologia , Transcriptase Reversa do HIV/metabolismo , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/fisiologia , Latência Viral/efeitos dos fármacos , Animais , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Cinética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Macaca , Masculino , RNA Viral/metabolismo , Vírus da Imunodeficiência Símia/enzimologia , Vírus da Imunodeficiência Símia/genética , Viremia/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Hepatitis C virus (HCV) RNA replication involves complex interactions among the 3'x RNA element within the HCV 3' untranslated region, viral and host proteins. However, many of the host proteins remain unknown. In this study, we devised an RNA affinity chromatography /2D/MASS proteomics strategy and identified nine putative 3' X-associated host proteins; among them is oxysterol-binding protein-related protein 4 (ORP4), a cytoplasmic receptor for oxysterols. We determined the relationship between ORP4 expression and HCV replication. A very low level of constitutive ORP4 expression was detected in hepatocytes. Ectopically expressed ORP4 was detected in the endoplasmic reticulum and inhibited luciferase reporter gene expression in HCV subgenomic replicon cells and HCV core expression in JFH-1-infected cells. Expression of ORP4S, an ORP4 variant that lacked the N-terminal pleckstrin-homology domain but contained the C-terminal oxysterol-binding domain also inhibited HCV replication, pointing to an important role of the oxysterol-binding domain in ORP4-mediated inhibition of HCV replication. ORP4 was found to associate with HCV NS5B and its expression led to inhibition of the NS5B activity. ORP4 expression had little effect on intracellular lipid synthesis and secretion, but it induced lipid droplet formation in the context of HCV replication. Taken together, these results demonstrate that ORP4 is a negative regulator of HCV replication, likely via interaction with HCV NS5B in the replication complex and regulation of intracellular lipid homeostasis. This work supports the important role of lipids and their metabolism in HCV replication and pathogenesis.
Assuntos
Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/virologia , Metabolismo dos Lipídeos , Receptores de Esteroides/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Linhagem Celular , Expressão Gênica , Hepatite C/genética , Hepatócitos/metabolismo , Humanos , Espaço Intracelular/metabolismo , Lipídeos/biossíntese , Ligação Proteica , Transporte Proteico , Receptores de Esteroides/genéticaRESUMO
The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (2012) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset contained potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with K(d)s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC(50)=5±2µM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.
Assuntos
Antivirais/farmacologia , DNA Helicases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Serina Endopeptidases/metabolismo , Tiazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Linhagem Celular , Fluorescência , Hepacivirus/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Ligação Proteica , Tiazóis/químicaRESUMO
A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 µM, inhibited the subgenomic HCV replicon at 10 µM, and was not toxic at 100 µM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 µM.
Assuntos
Antivirais/farmacologia , Hepacivirus/enzimologia , RNA Helicases/antagonistas & inibidores , Tiazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/isolamento & purificação , Benzotiazóis , Proteínas Sanguíneas/metabolismo , Linhagem Celular Tumoral , Corantes/química , Corantes/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Humanos , Camundongos , Polímeros , Ligação Proteica , RNA Viral/metabolismo , Replicon , Solubilidade , Relação Estrutura-Atividade , Tiazóis/química , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Viruses interact with and exploit the host cellular machinery for their multiplication and propagation. The MEK/ERK signaling pathway positively regulates replication of many RNA viruses. However, whether and how this signaling pathway affects hepatitis C virus (HCV) replication and production is not well understood. METHODS AND RESULTS: In this study, we took advantage of two well-characterized MEK/ERK inhibitors and MEK/ERK dominant negative mutants and investigated the roles of the MEK/ERK signaling pathway in HCV gene expression and replication. We showed that inhibition of MEK/ERK signaling enhanced HCV gene expression, plus- and minus-strand RNA synthesis, and virus production. In addition, we showed that this enhancement was independent of interferon-alpha (IFN-alpha) antiviral activity and did not require prior activation of the MEK/ERK signaling pathway. Furthermore, we showed that only MEK and ERK-2 but not ERK-1 was involved in HCV replication, likely through regulation of HCV RNA translation. CONCLUSIONS: Taken together, these results demonstrate a negative regulatory role of the MEK/ERK signaling pathway in HCV replication and suggest a potential risk in targeting this signaling pathway to treat and prevent neoplastic transformation of HCV-infected liver cells.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Viral da Expressão Gênica , Hepacivirus/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Regulação para Cima , Replicação Viral , Antivirais/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Interferon-alfa/metabolismo , Fígado/metabolismo , Biossíntese de Proteínas , RNA Viral/metabolismo , Transdução de SinaisRESUMO
Invention of subgenomic HCV replicon a few years ago and recent success of in vitro production of infectious HCV have improved our knowledge of the HCV life cycle, replication, pathogenesis, and screening of anti-HCV therapeutics. However, the highly genotype-dependent nature of the in vitro HCV production system has limited its potential for HCV research. In this study, we constructed a recombinant DNA-based HCV system that contained EF-1alpha promoter-driven HCV genotype 1b with HCV E1/E2 deleted and replaced by GFP. We co-transfected this recombinant cDNA with HCV E1/E2 or VSV-G expression plasmid into 293T cells, and we showed HCV protein expression and processing and demonstrated production of HCV-like particles in culture supernatant of co-transfected cells. These results support potential use of this system for studies on expression and processing of the HCV polyprotein and assembly and release of HCV-like particles.
Assuntos
DNA Complementar/genética , Hepacivirus/fisiologia , Rim/metabolismo , Rim/virologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Vírion/metabolismo , Linhagem Celular , Vetores Genéticos/genética , Humanos , Vírion/genéticaRESUMO
Hepatitis C virus (HCV) is a blood-borne pathogen that poses a significant threat to public health worldwide. The genetic diversity and distribution of HCV genotypes in non-Western countries, particularly subSaharan Africa, is poorly documented. This study reports a phylogenetic analysis of core and NS5B gene sequences of 37 HCV strains sampled in Cameroon. A high level of genetic diversity of both genotypes 1 and 4 was found, indicating a unique pattern of long-term HCV infection that has not been observed elsewhere. These results lead to the hypothesis that these HCV genotypes originated and diversified in west Central Africa before spreading to other regions.
Assuntos
Doenças Endêmicas , Hepacivirus/genética , Hepatite C/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Camarões/epidemiologia , Feminino , Variação Genética , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Hepatite C/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Parenteral transmission of HCV is well established but other possible routes such as heterosexual transmission are still questioned. The Central African region is characterised by a high HCV endemicity without any evidence on the route of transmission. The information on HCV genotypes that circulate in this area is also limited and controversial. HIV infection is very frequent in this region and mostly acquired via the heterosexual route. The aim of this work was to investigate the trend of HCV infection and genotypes among HIV high-risk groups from Cameroon. Four hundred eighty-two patients including 229 tuberculosis clinic attendants, 184 sexually transmitted disease clinic attendants, and 69 HIV clinical suspects from another clinic were enrolled. All plasma samples were screened for antibodies to HCV and HCV RNA. Genotypes were assigned by sequencing a 5'UTR amplified fragment. The overall prevalence of HCV markers was 11.6% and a significant increasing trend with respect to age was observed. A proportion of 64.1% (34/53) of HCV antibody positive samples was viraemic. HCV RNA was found in 3 samples that were indeterminate in RIBA 3.0. One was negative in the antibody screening test and the two others were weakly positive. The data on HCV genotypes revealed that genotype 1 was involved in 57% of viraemias, genotype 2 in 24%, while genotypes 4 and 5a accounted for 16 and 3%, respectively. In contrary to the predominance of genotype 4 reported in some African countries and even the neighbouring countries, these data demonstrate clearly that HCV infection in Cameroon is dominated by genotypes 1 and 2. No association was found between the HCV markers and the presence of HIV infection. It is concluded, therefore, that the heterosexual route plays a minor role in HCV transmission in this country.