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1.
Proc Natl Acad Sci U S A ; 121(5): e2308776121, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38252831

RESUMO

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.


Assuntos
Influenza Humana , Vírus , Animais , Camundongos , Humanos , Proteoma , Peptídeos/farmacologia , Descoberta de Drogas
2.
Int J Mol Sci ; 23(2)2022 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-35055105

RESUMO

Hepatitis C virus (HCV) relies on cellular lipid metabolism for its replication, and actively modulates lipogenesis and lipid trafficking in infected hepatocytes. This translates into an intracellular accumulation of triglycerides leading to liver steatosis, cirrhosis and hepatocellular carcinoma, which are hallmarks of HCV pathogenesis. While the interaction of HCV with hepatocyte metabolic pathways is patent, how viral proteins are able to redirect central carbon metabolism towards lipogenesis is unclear. Here, we report that the HCV protein NS5A activates the glucokinase (GCK) isoenzyme of hexokinases through its D2 domain (NS5A-D2). GCK is the first rate-limiting enzyme of glycolysis in normal hepatocytes whose expression is replaced by the hexokinase 2 (HK2) isoenzyme in hepatocellular carcinoma cell lines. We took advantage of a unique cellular model specifically engineered to re-express GCK instead of HK2 in the Huh7 cell line to evaluate the consequences of NS5A-D2 expression on central carbon and lipid metabolism. NS5A-D2 increased glucose consumption but decreased glycogen storage. This was accompanied by an altered mitochondrial respiration, an accumulation of intracellular triglycerides and an increased production of very-low density lipoproteins. Altogether, our results show that NS5A-D2 can reprogram central carbon metabolism towards a more energetic and glycolytic phenotype compatible with HCV needs for replication.


Assuntos
Glucoquinase/metabolismo , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Glicogênio/metabolismo , Glicólise , Interações Hospedeiro-Patógeno , Humanos , Metabolismo dos Lipídeos , Lipogênese , Mitocôndrias/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química
3.
J Viral Hepat ; 28(12): 1690-1698, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34467593

RESUMO

The nuclear farnesoid X receptor (FXR) regulates bile acid homeostasis and is a drug target for metabolic liver diseases. FXR also plays an important role in hepatitis B virus (HBV) DNA transcription. In vitro and in mice, FXR agonist treatment leads to inhibition of viral replication and a decline in viral proteins, pregenomic RNA (pgRNA) and HBV DNA levels. We aimed to translate this to a clinical use by primarily evaluating the safety and secondary the anti-viral effect of Vonafexor, a FXR agonist, in chronic hepatitis B (CHB) patients. In total, 73 CHB patients were enrolled in a two-part Phase Ib double-blind, placebo-controlled trial. Patients were randomized to receive oral Vonafexor (100, 200 and 400 mg once daily, or 200 mg twice daily), placebo, or entecavir (Part A, n = 48) or to receive Vonafexor (300 mg once daily or 150 mg twice daily), or placebo, combined with pegylated-interferon-α2a (Part B, n = 25) for 29 days. Patients were followed up for 35 days. Enrolled CHB patients were mostly HBeAg-negative. Vonafexor was overall well tolerated and safe. The most frequent adverse events were moderate gastrointestinal events. Pruritus was more frequent with twice-daily compared with once-daily regimens (56%-67% vs. 16%, respectively, p < 0.05). Vonafexor monotherapy of 400 mg once daily decreased HBsAg concentrations (-0.1 log10  IU/mL, p < 0.05), and Vonafexor/pegylated-IFN-α2a combination therapy decreased HBcrAg and pgRNA. In conclusion, Vonafexor was safe with a decline in HBV markers observed in CHB patients suggesting a potential anti-viral effect the therapeutic potential of which has to be evaluated in larger trials.


Assuntos
Hepatite B Crônica , Preparações Farmacêuticas , Antivirais/efeitos adversos , DNA Viral , Antígenos de Superfície da Hepatite B , Antígenos E da Hepatite B , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Humanos
4.
J Transl Med ; 18(1): 319, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811513

RESUMO

In less than 20 years, three deadly coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have emerged in human population causing hundreds to hundreds of thousands of deaths. Other coronaviruses are causing epizootic representing a significant threat for both domestic and wild animals. Members of this viral family have the longest genome of all RNA viruses, and express up to 29 proteins establishing complex interactions with the host proteome. Deciphering these interactions is essential to identify cellular pathways hijacked by these viruses to replicate and escape innate immunity. Virus-host interactions also provide key information to select targets for antiviral drug development. Here, we have manually curated the literature to assemble a unique dataset of 1311 coronavirus-host protein-protein interactions. Functional enrichment and network-based analyses showed coronavirus connections to RNA processing and translation, DNA damage and pathogen sensing, interferon production, and metabolic pathways. In particular, this global analysis pinpointed overlooked interactions with translation modulators (GIGYF2-EIF4E2), components of the nuclear pore, proteins involved in mitochondria homeostasis (PHB, PHB2, STOML2), and methylation pathways (MAT2A/B). Finally, interactome data provided a rational for the antiviral activity of some drugs inhibiting coronaviruses replication. Altogether, this work describing the current landscape of coronavirus-host interactions provides valuable hints for understanding the pathophysiology of coronavirus infections and developing effective antiviral therapies.


Assuntos
Infecções por Coronavirus/metabolismo , Coronavirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Mapas de Interação de Proteínas , Proteínas Virais/metabolismo , Animais , Betacoronavirus/fisiologia , COVID-19 , Coronavirus/química , Infecções por Coronavirus/virologia , Bases de Dados de Proteínas , Humanos , Proteínas Mitocondriais/metabolismo , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Proibitinas , SARS-CoV-2 , Fatores de Transcrição/metabolismo , Replicação Viral/genética
5.
FASEB J ; 33(2): 2472-2483, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30307769

RESUMO

Hepatitis B virus (HBV) infection and bile acid (BA) metabolism are interdependent: infection modifies the expression of the BA nuclear receptor farnesoid X receptor (FXR)-α, and modulation of FXRα activity by ligands alters HBV replication. Mechanisms of HBV control by FXRα remain to be unveiled. FXRα silencing in HBV-infected HepaRG cells decreased the viral covalently closed circular (ccc)DNA pool size and transcriptional activity. Treatment with the FXRα agonist GW4064 inhibited FXRα proviral effect on cccDNA similarly for wild-type and hepatitis B viral X protein (HBx)-deficient virus, whereas agonist-induced inhibition of pregenomic and precore RNA transcription and viral DNA secretion was HBx dependent. These data indicated that FXRα acts as a proviral factor by 2 different mechanisms, which are abolished by FXRα stimulation. Finally, infection of C3H/HeN mice by a recombinant adeno-associated virus-2/8-HBV vector induced a sustained HBV replication in young mice in contrast with the transient decline in adult mice. Four-week GW4064 treatment of infected C3H/HeN mice decreased secretion of HBV DNA and HB surface antigen in adult mice only. These results suggest that the physiologic balance of FXRα expression and activation by bile acid is a key host metabolic pathway in the regulation of HBV infection and that FXRα can be envisioned as a target for HBV treatment.-Mouzannar, K., Fusil, F., Lacombe, B., Ollivier, A., Ménard, C., Lotteau, V., Cosset, F.-L., Ramière, C., André, P. Farnesoid X receptor α is a proviral host factor for hepatitis B virus that is inhibited by ligands in vitro and in vivo.


Assuntos
Regulação da Expressão Gênica , Vírus da Hepatite B/patogenicidade , Hepatite B/virologia , Provírus/patogenicidade , Receptores Citoplasmáticos e Nucleares/metabolismo , Replicação Viral , Animais , DNA Viral/genética , Feminino , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/patologia , Vírus da Hepatite B/genética , Humanos , Técnicas In Vitro , Ligantes , Camundongos , Camundongos Endogâmicos C3H , Provírus/genética
6.
J Immunol ; 201(5): 1510-1521, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30037846

RESUMO

Cell metabolism now appears as an essential regulator of immune cells activation. In particular, TLR stimulation triggers metabolic reprogramming of dendritic cells (DCs) with an increased glycolytic flux, whereas inhibition of glycolysis alters their functional activation. The molecular mechanisms involved in the control of glycolysis upon TLR stimulation are poorly understood for human DCs. TLR4 activation of human monocyte-derived DCs (MoDCs) stimulated glycolysis with an increased glucose consumption and lactate production. Global hexokinase (HK) activity, controlling the initial rate-limiting step of glycolysis, was also increased. TLR4-induced glycolytic burst correlated with a differential modulation of HK isoenzymes. LPS strongly enhanced the expression of HK2, whereas HK3 was reduced, HK1 remained unchanged, and HK4 was not expressed. Expression of the other rate-limiting glycolytic enzymes was not significantly increased. Exploring the signaling pathways involved in LPS-induced glycolysis with various specific inhibitors, we observed that only the inhibitors of p38-MAPK (SB203580) and of HIF-1α DNA binding (echinomycin) reduced both the glycolytic activity and production of cytokines triggered by TLR4 stimulation. In addition, LPS-induced HK2 expression required p38-MAPK-dependent HIF-1α accumulation and transcriptional activity. TLR1/2 and TLR2/6 stimulation increased glucose consumption by MoDCs through alternate mechanisms that are independent of p38-MAPK activation. TBK1 contributed to glycolysis regulation when DCs were stimulated via TLR2/6. Therefore, our results indicate that TLR4-dependent upregulation of glycolysis in human MoDCs involves a p38-MAPK-dependent HIF-1α accumulation, leading to an increased HK activity supported by enhanced HK2 expression.


Assuntos
Células Dendríticas/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Hexoquinase/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Monócitos/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Células Cultivadas , Células Dendríticas/patologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/toxicidade , Monócitos/patologia , Estabilidade Proteica , Receptor 4 Toll-Like/agonistas
7.
Liver Transpl ; 24(8): 1091-1100, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29665189

RESUMO

The incidence and impact of anti-human leukocyte antigen donor-specific alloantibodies (DSAs) developing after liver transplantation (LT) remains controversial and not extensively studied. The aim of the present study was to assess the incidence of DSAs, to identify risk factors for the development of DSAs, and to understand the impact of DSAs in a large population of adult LT recipients. This single-center retrospective study included all adult patients who underwent a first LT between 2000 and 2010 in our center. The study population mainly consisted of male patients, the mean age was 52.4 years, and the main indication was alcoholic cirrhosis (54.1%). From the 297 patients included in the cross-sectional study, 14 (4.7%) had preformed DSAs, and 59 (19.9%) presented de novo DSAs (12.2% at 1 year, 13.4% at 5 years, and 19.5% at 10 years). Multivariate analysis found that female donor sex (hazard ratio [HR], 1.50; 95% confidence interval [CI], 1.12-2.01; P = 0.01) and delay between LT and DSA screening (HR, 1.10; 95% CI, 1.01-1.20; P = 0.03) were associated with occurrence of de novo DSAs. From the 190 patients included in the subgroup longitudinal analysis, exposure to tacrolimus (mean trough level during the periods 0-2 years and 0-3 years) was significantly lower for patients having DSAs at 5 years. Concerning histology, only acute rejection (P = 0.04) and portal fibrosis ≥2 (P = 0.02) were more frequent at 1 year for patients with DSAs. Patient survival and graft survival were not significantly different according to the presence or not of DSAs at 1 year. Among the 44 patients who had de novo or persistent preformed DSAs, the diagnosis of antibody-mediated rejection was made in 4 (9.1%) patients after 1, 47, 61, and 74 months following LT. In conclusion, the results of the present study suggest that DSAs are observed in a minority of LT adult patients, with limited overall impact on graft and patient outcome.


Assuntos
Rejeição de Enxerto/epidemiologia , Isoanticorpos/sangue , Cirrose Hepática Alcoólica/cirurgia , Transplante de Fígado/efeitos adversos , Complicações Pós-Operatórias/epidemiologia , Adulto , Estudos Transversais , Feminino , Seguimentos , Rejeição de Enxerto/sangue , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Antígenos HLA/imunologia , Humanos , Imunossupressores/uso terapêutico , Incidência , Isoanticorpos/imunologia , Isoanticorpos/isolamento & purificação , Cirrose Hepática Alcoólica/mortalidade , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/sangue , Complicações Pós-Operatórias/imunologia , Complicações Pós-Operatórias/prevenção & controle , Prevalência , Estudos Retrospectivos , Fatores de Risco , Análise de Sobrevida , Tacrolimo/uso terapêutico , Transplantados , Resultado do Tratamento
8.
Angew Chem Int Ed Engl ; 57(17): 4787-4791, 2018 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-29457857

RESUMO

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native-like lipid environment. Cell-free protein synthesis and solid-state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell-free expression, and that the protein self-assembles into subviral particles. Proton-detected 2D NMR spectra recorded at a magic-angle-spinning frequency of 110 kHz on <500 µg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.


Assuntos
Vírus da Hepatite B/química , Ressonância Magnética Nuclear Biomolecular , Proteínas da Matriz Viral/química , Sistema Livre de Células , Conformação Proteica
9.
J Med Virol ; 89(11): 1912-1919, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28590068

RESUMO

Sanger population sequencing (SPS) is the reference technique to monitor HIV-1-infected patients' therapy. Ultra-deep sequencing (UDS), which allows quantitative detection of drug resistance mutations, may be an alternative method. The study aimed to compare reproducibility and predictions of UDS versus SPS in a routine setting. A control containing low-abundance variants was repeatedly tested and clinical plasma samples from 100 patients were prospectively assayed by SPS and UDS using the Roche 454 system. Complete analysis by UDS was available for 88% of samples with various viral loads and subtypes. Comparison of detection thresholds found that SPS sensitivity was variable. Variations found by UDS between 5% to >20% were detected by SPS in 25% to more than 80% of samples. At the 5% cut-off, disagreements were rare and in most cases UDS detected an additional protease secondary mutation, suggesting a possible resistance to a protease inhibitor according to the 2015 ANRS algorithm. Mutations found on reverse transcriptase by only UDS were often explained by previous therapy. UDS with a variant detection threshold at 5% might allow therapy management with minimal differences compared to population sequencing while providing additional information for further determination of pertinent cutoff values for specific resistance mutations.


Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Fármacos Anti-HIV/uso terapêutico , Feminino , Genótipo , Técnicas de Genotipagem , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Masculino , Reprodutibilidade dos Testes , Software , Carga Viral/efeitos dos fármacos
10.
FASEB J ; 30(9): 3146-54, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27251172

RESUMO

Hepatitis B virus (HBV) and bile salt metabolism seem tightly connected. HBV enters hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP), the genome of which contains 2 active farnesoid X receptor (FXR) α response elements that participate in HBV transcriptional activity. We investigated in differentiated HepaRG cells and in primary human hepatocytes (PHHs) effects of FXR activation on HBV replication and of infection on the FXR pathway. In HepaRG cells, FXR agonists (6-ethyl chenodeoxycholic acid and GW4064), but no antagonist, and an FXR-unrelated bile salt inhibited viral mRNA, DNA, and protein production (IC50, 0.1-0.5 µM) and reduced covalently closed circular DNA pool size. These effects were independent of the NTCP inhibitor cyclosporine-A, which suggests inhibition occurred at a postentry step. Similar results were obtained in PHHs with GW4064. Infection of these cells increased expression of FXR and modified expression of FXR-regulated genes SHP, APOA1, NTCP, CYP7A1, and CYP8B1 with a more pronounced effect in PHHs than in HepaRG cells. FXR agonists reversed all but one of the HBV-induced FXR gene profile modifications. HBV replication and FXR regulation seem to be interdependent, and altered bile salt metabolism homeostasis might contribute to the persistence of HBV infection.-Radreau, P., Porcherot, M., Ramière, C., Mouzannar, K., Lotteau, V., André, P. Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.


Assuntos
Diferenciação Celular/fisiologia , Vírus da Hepatite B/fisiologia , Hepatócitos/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Replicação Viral/fisiologia , Linhagem Celular , DNA Viral , Regulação da Expressão Gênica/fisiologia , Humanos , RNA Viral , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Replicação Viral/efeitos dos fármacos
11.
Phytochem Anal ; 28(1): 42-49, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27976468

RESUMO

INTRODUCTION: The "Jardin de Granville" modern rose variety not only combines the morphological properties of its two parental cultivars, but also possesses better agronomic characteristics (abundant blooms, strong growth and vitality, high resistance to common rose diseases). In addition, it shows remarkable biological properties such as a high ability to decrease inflammatory and oxidative stress on skin cells. That is why Parfums Christian Dior selected this rose variety to be an active ingredient in luxury cosmetics. OBJECTIVES: To identify the characteristic molecular signature of "Jardin de Granville" compared with its parents "Annapurna" and "John Clare", by the mean of a non-targeted metabolomic comparison. MATERIAL AND METHODS: Wood, flower and leaf hydro-alcoholic extracts were analysed by UHPLC-ESI-HRMS. The fingerprints were then submitted to unsupervised multivariate analyses involving principal component analysis (PCA) and hierarchical ascendant classification (HAC). Analysis of variance (ANOVA) was finally performed to highlight the significant differences in each group of organs. RESULTS: The extracts were composed of phenolic compounds such as hydrolysable and condensed tannins and flavonol derivatives. Three groups of extracts were clustered as a function of the variety. The compounds overexpressed in "Jardin de Granville" variety were highlighted thanks to ANOVA test. Flower was the most discriminative organ with 15 overexpressed molecules. Auto MS/MS analyses led to their tentative identifications. CONCLUSION: The non-targeted metabolomic approach revealed the importance of tannins to discriminate close rose varieties. The overexpressed hydrolysable tannins characteristic of "Jardin de Granville" can be responsible for the antioxidant and anti-inflammatory properties of the rose cosmetic ingredients. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Rosa/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Metabolômica , Análise Multivariada , Reprodutibilidade dos Testes
12.
J Hepatol ; 65(3): 499-508, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27174035

RESUMO

BACKGROUND & AIMS: Ribavirin monotherapy is the preferred treatment for chronic hepatitis E, although occasional treatment failure occurs. We present a patient with chronic hepatitis E experiencing ribavirin treatment failure with a completely resistant phenotype. We aimed to identify viral mutations associated with treatment failure and explore the underlying mechanisms. METHODS: Viral genomes were deep-sequenced at different time points and the role of identified mutations was assessed in vitro using mutant replicons, antiviral assays, cell culture of patient-derived virus and deep-sequencing. RESULTS: Ribavirin resistance was associated with Y1320H, K1383N and G1634R mutations in the viral polymerase, but also an insertion in the hypervariable region comprising a duplication and a polymerase-derived fragment. Analysis of these genome alterations in vitro revealed replication-increasing roles for Y1320H and G1634R mutations and the hypervariable region insertion. In contrast, the K1383N mutation in the polymerase F1-motif suppressed viral replication and increased the in vitro sensitivity to ribavirin, contrary to the clinical phenotype. Analysis of the replication of mutant full-length virus and in vitro culturing of patient-derived virus confirmed that sensitivity to ribavirin was retained. Finally, deep-sequencing of hepatitis E virus genomes revealed that ribavirin is mutagenic to viral replication in vitro and in vivo. CONCLUSIONS: Mutations Y1320H, G1634R and the hypervariable region insertion compensated for K1383N-associated replication defects. The specific role of the K1383N mutation remains enigmatic, but it appears to be of importance for the ribavirin resistant phenotype in this patient. LAY SUMMARY: Ribavirin is the most common treatment for chronic hepatitis E and is mostly effective, although some cases of ribavirin treatment failure have been described. Here, we report on a particular case of ribavirin resistance and investigate the underlying causes of treatment failure. Mutations in the viral polymerase, an essential enzyme for viral replication, appear to be responsible.


Assuntos
Vírus da Hepatite E , Antivirais , Farmacorresistência Viral , Humanos , Mutação , Ribavirina , Falha de Tratamento , Replicação Viral
13.
Arch Virol ; 161(12): 3495-3507, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27659676

RESUMO

Human immunodeficiency virus type 1 (HIV-1) unspliced mRNA drives the expression of both Gag and Gag-Pol polyproteins by using both cap- and internal ribosome entry site (IRES)-dependent translation initiation mechanisms. An IRES has been described in the matrix coding region that is involved in the production of shorter isoforms of Gag. However, up to now, this has only been shown with sequences derived from the HIV-1 laboratory strains (NL4.3 and HXB2) and never from clinical HIV-1 isolates. We have isolated ~70 sequences from HIV-1-positive patients that we have sequenced and cloned into an expression vector to monitor their ability to drive translation of Gag p55 and the shorter isoforms both in vitro and ex vivo. The results indicate that (1) the translational efficiency from the AUG-p55 varies significantly among the different isolates; (2) expression initiated at AUG-p40 codon is independent of translation initiation at the AUG-p55 triplet; and (3) all sequences promote expression of shorter Gag isoforms, in particular in Jurkat T cells, in which internal initiation occurs exclusively and directly at the AUG-p40 codon. The composition of the first ~800 nucleotides of the HIV-1 unspliced mRNA modulates the expression initiated both at the AUG-p55 and AUG-p40 codons and may impact viral production and replication. Interestingly, the AUG-p40 codon and its surrounding nucleotide context are conserved amongst clinical isolates and are used as a translation initiation site to produce a shorter Gag isoform.


Assuntos
Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Isoformas de Proteínas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Clonagem Molecular , Feminino , Expressão Gênica , Humanos , Células Jurkat , Masculino , Biossíntese de Proteínas , RNA Viral/genética , Análise de Sequência de DNA
14.
J Virol ; 88(6): 3246-54, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24390321

RESUMO

UNLABELLED: The study of cellular central carbon metabolism modulations induced by viruses is an emerging field. Human cytomegalovirus (HCMV), herpes simplex virus (HSV), Kaposi's sarcoma-associated herpesvirus (KSHV), and hepatitis C virus (HCV) have been shown recently to reprogram cell metabolism to support their replication. During HCV infection the global glucidolipidic metabolism of hepatocytes is highly impacted. It was suggested that HCV might modify glucose uptake and glycolysis to increase fatty acids synthesis, but underlying mechanisms have not been completely elucidated. We thus investigated how HCV may modulate glycolysis. We observed that in infected Huh7.5 cells and in subgenomic replicon-positive Huh9.13 cells, glucose consumption as well as lactate secretion was increased. Using protein complementation assays and coimmunoprecipitation, we identified a direct interaction between the HCV NS5A protein and cellular hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis. NS5A expression was sufficient to enhance glucose consumption and lactate secretion in Huh7.5 cells. Moreover, determination of HK activity in cell homogenates revealed that addition of exogenous NS5A protein, either the full-length protein or its D2 or D3, but not D1, domain, was sufficient to increase enzyme activity. Finally, determination of recombinant HK2 catalytic parameters (V(max) and K(m)) in the presence of NS5A identified this viral protein as an activator of the enzyme. In summary, this study describes a direct interaction between HCV NS5A protein and cellular HK2 which is accompanied by an increase in HK2 activity that might contribute to an increased glycolysis rate during HCV infection. IMPORTANCE: Substantial evidence indicates that viruses reprogram the central carbon metabolism of the cell to support their replication. Nevertheless, precise underlying mechanisms are poorly described. Metabolic pathways are structured as connected enzymatic cascades providing elemental biomolecular blocks necessary for cell life and viral replication. In this study, we observed an increase in glucose consumption and lactate secretion in HCV-infected cells, revealing higher glycolytic activity. We also identified an interaction between the HCV NS5A nonstructural protein and cellular hexokinase 2, the first rate-limiting enzyme of glycolysis. This interaction results in an enhancement of catalytic parameters of the enzyme, which might explain, at least in part, the aerobic glycolysis shift observed in HCV-infected cells.


Assuntos
Hepacivirus/enzimologia , Hepatite C/enzimologia , Hexoquinase/metabolismo , Regulação para Cima , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular , Glucose/metabolismo , Glicólise , Hepacivirus/genética , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/virologia , Hexoquinase/genética , Humanos , Ligação Proteica , Proteínas não Estruturais Virais/genética
15.
PLoS Pathog ; 9(7): e1003440, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23853584

RESUMO

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.


Assuntos
Adenosina Desaminase/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Replicação Viral , Adenosina Desaminase/química , Adenosina Desaminase/genética , Transporte Biológico , Linhagem Celular , Vírus da Dengue/enzimologia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade da Espécie , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genética
16.
FASEB J ; 28(3): 1454-63, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24297698

RESUMO

Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.


Assuntos
Vírus da Hepatite B/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Sirtuína 1/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Primers do DNA , Humanos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Interferente Pequeno
17.
EMBO Rep ; 14(10): 938-44, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24008843

RESUMO

Virus-host interactomes are instrumental to understand global perturbations of cellular functions induced by infection and discover new therapies. The construction of such interactomes is, however, technically challenging and time consuming. Here we describe an original method for the prediction of high-confidence interactions between viral and human proteins through a combination of structure and high-quality interactome data. Validation was performed for the NS1 protein of the influenza virus, which led to the identification of new host factors that control viral replication.


Assuntos
Interações Hospedeiro-Patógeno , Modelos Biológicos , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cães , Humanos , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Orthomyxoviridae/metabolismo , Orthomyxoviridae/fisiologia , Ligação Proteica , Proteínas não Estruturais Virais/química , Replicação Viral
18.
Phytochem Anal ; 26(3): 189-201, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25645670

RESUMO

INTRODUCTION: A non-targeted approach to characterise the phytochemical composition of the flower organ of an original rose cultivar 'Jardin de Granville'® was developed. Particular attention was paid to the less documented molecular families of intermediate polarity, compared with the polyphenol family (anthocyanins, flavonoids, tannins) and volatile compounds. OBJECTIVE: To develop a molecular fingerprinting method for the rapid qualitative phytochemical characterisation of the rose flower ethyl acetate extract. MATERIAL AND METHODS: An ultra-HPLC with atmospheric pressure photoionisation (APPI) and quadrupole time-of-flight (QTOF) MS/MS combined with microwave-assisted extraction was carried out for ethyl acetate extracts as an intermediate polarity extraction solvent in order to obtain the most exhaustive extract containing a large range of molecular families. An optimised methodology based on the coupling of the UHPLC and APPI source with a QTOF analyser was developed to characterise the extracted molecules. RESULTS: Sixty-one compounds were identified in the extract, covering eight molecular families of intermediate polarity ranging from polyphenols to triglycerides. The presence of flavonoids with anti-oxidant properties and of triterpenoids with potential anti-inflammatory activity was evidenced and cell-wall constituents such as fatty acids, glycolipids, sphingolipids and acylated sterol glycosides were characterised. Some chlorophyll derivatives were also detected. CONCLUSION: The method developed is appropriate for fast phytochemical evaluation of rose ethyl acetate extract. It produced accurate mass and MS/MS spectra, which permitted identification of a wide range of compounds of intermediate polarity.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Flores/química , Extratos Vegetais/análise , Rosa/química , Espectrometria de Massas em Tandem/métodos , Acetatos/química , Pressão Atmosférica , Cromatografia Líquida de Alta Pressão/normas , Ácidos Graxos/análise , Ácidos Graxos/química , Flavonoides/química , Glicosídeos/análise , Glicosídeos/química , Fenóis/análise , Fenóis/química , Fitosteróis/análise , Fitosteróis/química , Extratos Vegetais/química , Taninos/análise , Taninos/química , Triterpenos/análise , Triterpenos/química
19.
Int J Mol Sci ; 16(9): 22223-42, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26389885

RESUMO

Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724-1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC.


Assuntos
Técnicas de Genotipagem/métodos , Hepatite C Crônica/genética , Interleucinas/genética , Lipase/genética , Proteínas de Membrana/genética , Desnaturação de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Interferons , Masculino , Pessoa de Meia-Idade
20.
FASEB J ; 27(10): 4027-40, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23781096

RESUMO

TGF-ß signaling induces epithelial to mesenchymal transition (EMT) and plays an important role in hepatocellular carcinoma (HCC) development. Clinical observations indicate that hepatitis C virus (HCV) chronic infection, which is a major cause of HCC, induces TGF-ß signaling perturbations. Here, we investigate the mechanisms by which HCV nonstructural proteins interfere with TGF-ß signaling, in human hepatoma cell lines expressing HCV subgenomic replicon. A transcriptomic study showed that TGF-ß stimulation of these cells resulted in a protumoral gene expression profile and in up-regulation of EMT-related genes compared to control interferon-treated cells not expressing HCV proteins. We found that the viral protease NS3-4A interacted with SMURF2, a negative regulator of TGF-ß signaling. In cells expressing HCV subgenomic replicon or NS3-4A, TGF-ß stimulation induced an increased expression of SMAD-dependent genes compared to control cells. This enhanced signaling was suppressed by SMURF2 overexpression and mimicked by SMURF2 silencing. In addition, NS3-4A expression resulted in an increased and prolonged TGF-ß-induced phosphorylation of SMAD2/3 that was abrogated by SMURF2 overexpression. Neither NS3-4A protease activity nor SMURF2 ubiquitin-ligase activity was required to affect TGF-ß signaling. Therefore, by targeting SMURF2, NS3-4A appears to block the negative regulation of TGF-ß signaling, increasing the responsiveness of cells to TGF-ß.


Assuntos
Hepacivirus/metabolismo , Peptídeo Hidrolases/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/fisiologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Peptídeo Hidrolases/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Ubiquitina-Proteína Ligases/genética
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