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2.
Nat Commun ; 14(1): 433, 2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36702826

RESUMO

Hepatitis C virus (HCV) uses a hybrid entry mechanism. Current structural data suggest that upon exposure to low pH and Cluster of Differentiation 81 (CD81), the amino terminus of envelope glycoprotein E2 becomes ordered and releases an internal loop with two invariant aromatic residues into the host membrane. Here, we present the structure of an amino-terminally truncated E2 with the membrane binding loop in a bent conformation and the aromatic side chains sequestered. Comparison with three previously reported E2 structures with the same Fab indicates that this internal loop is flexible, and that local context influences the exposure of hydrophobic residues. Biochemical assays show that the amino-terminally truncated E2 lacks the baseline membrane-binding capacity of the E2 ectodomain. Thus, the amino terminal region is a critical determinant for both CD81 and membrane interaction. These results provide new insights into the HCV entry mechanism.


Assuntos
Hepacivirus , Hepatite C , Humanos , Hepacivirus/metabolismo , Ligação Proteica , Proteínas do Envelope Viral/metabolismo , Tetraspanina 28/química , Tetraspanina 28/metabolismo
3.
Proc Natl Acad Sci U S A ; 120(1): e2211425120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36577062

RESUMO

De novo viral RNA-dependent RNA polymerases (RdRPs) utilize their priming element (PE) to facilitate accurate initiation. Upon transition to elongation, the PE has to retreat from the active site to give room to the template-product RNA duplex. However, PE conformational change upon this transition and the role of PE at elongation both remain elusive. Here, we report crystal structures of RdRP elongation complex (EC) from dengue virus serotype 2 (DENV2), demonstrating a dramatic refolding of PE that allows establishment of interactions with the RNA duplex backbone approved to be essential for EC stability. Enzymology data from both DENV2 and hepatitis C virus (HCV) RdRPs suggest that critical transition of the refolding likely occurs after synthesis of a 4- to 5-nucleotide (nt) product together providing a key basis in understanding viral RdRP transition from initiation to elongation.


Assuntos
RNA Polimerase Dependente de RNA , RNA , RNA Polimerase Dependente de RNA/metabolismo , Hepacivirus/metabolismo , Domínio Catalítico , Nucleotídeos , RNA Viral/genética
4.
Sci Rep ; 12(1): 21769, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36526719

RESUMO

Although people all know that nicotine in tobacco smoke is the key to cause health damage, they ignore the synergistic effect of a large number of Volatile Organic Compounds (VOCs) produced by incomplete tobacco combustion on nicotine or cotinine metabolism. Our aim is to investigate the association between serum VOCs and cotinine in smokers infected with HIV, HBV or HCV. National Health and Nutrition Examination Survey (NHANES 2005-2018) database, including 13,652 nationally representative subjects' sociodemographic characteristics and serological indicators, was used in this study. Smokers living with human immunodeficiency virus (HIV), hepatitis B virus (HBV) or hepatitis C virus (HCV) were compared to non-infected population. The correlation between VOCs and cotinine as well as the effects of VOCs on cotinine metabolism were analyzed by Spearman correlation analysis and multivariable logistic regression analysis, respectively. Among HIV, HBV, or HCV infected smokers with the largest exposure dose to tobacco, the intensity of the association between VOCs and cotinine was the strongest. The results of multivariable binary logistic regression showed that high concentrations of 1,2-Dichlorobenzene (OR:1.036, CI:1.009-1.124), Benzene (OR:1.478, CI:1.036-2.292), Carbon Tetrachloride (OR:1.576, CI:1.275-2.085) and 2,5-Dimethylfuran (OR:1.091, CI:1.030-1.157) in blood might be independent risk factors leading to the increase of serum metabolite cotinine in smokers.


Assuntos
Infecções por HIV , Hepatite C , Poluição por Fumaça de Tabaco , Compostos Orgânicos Voláteis , Adulto , Humanos , Cotinina/análise , Vírus da Hepatite B/metabolismo , Compostos Orgânicos Voláteis/análise , Inquéritos Nutricionais , Hepacivirus/metabolismo , Nicotina/análise , Poluição por Fumaça de Tabaco/efeitos adversos , HIV/metabolismo , Hepatite C/epidemiologia , Infecções por HIV/epidemiologia
5.
J Mol Model ; 29(1): 25, 2022 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-36580076

RESUMO

CONTEXT: Egypt has a high prevalence of the hepatitis C virus (HCV) genotype 4a (GT-4a). Unfortunately, the high resistance it exhibited still was not given the deserved attention in the scientific community. There is currently no consensus on the NS5A binding site because the crystal structure of HCV NS5A has not been resolved. The prediction of the binding modes of direct-acting antivirals (DAA) with the NS5A is a point of controversy due to the fact that several research groups presented different interaction models to elucidate the NS5A binding site. Consequently, a 3D model of HCV NS5A GT-4a was constructed and evaluated using molecular dynamics (MD) simulations. The generated model implies an intriguing new orientation of the AH relative to domain I. Additionally, the probable binding modes of marketed NS5A inhibitors were explored. MD simulations validated the stability of the predicted protein-ligand complexes. The suggested model predicts that daclatasvir and similar drugs bind symmetrically to HCV NS5A GT-4a. This will allow for the development of new NS5A-directed drugs, which may result in reduced resistance and/or a wider range of effectiveness against HCV. METHODS: The 3D model of HCV NS5A GT-4a was constructed using the comparative modeling approach of the web-based application Robetta. Its stability was tested with 200-ns MD simulations using the Desmond package of Schrodinger. The OPLS2005 force field was assigned for minimization, and the RMSD, RMSF, and rGyr were tracked throughout the MD simulations. Fpocket was used to identify druggable protein pockets (cavities) over the simulation trajectories. The binding modes of marketed NS5A inhibitors were then generated and refined with the aid of docking predictions made by FRED and AutoDock Vina. The stability of these drugs in complex with GT-4a was investigated by using energetic and structural analyses over MD simulations. The Prime MM-GBSA (molecular mechanics/generalized Born surface area) method was used as a validation tool after the docking stage and for the averaged clusters after the MD simulation stage. We utilized PyMOL and VMD to visualize the data.


Assuntos
Antivirais , Hepatite C Crônica , Humanos , Antivirais/química , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C Crônica/tratamento farmacológico , Imidazóis/farmacologia , Imidazóis/química , Genótipo , Proteínas não Estruturais Virais/química , Farmacorresistência Viral/genética
6.
Sci Rep ; 12(1): 19528, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376416

RESUMO

NOD-like receptor pyrin domain containing 3 (NLRP3) is a microbial and danger signal sensor that acts as a regulator of inflammation via activation of Caspase-1 (CASP1) and has been identified as a major contributor to human liver diseases. The present study was conducted to investigate the association between NLRP3 and the progression of hepatitis C virus (HCV)-related liver disease. Serum NLRP3 levels were analyzed in 49 patients with chronic HCV infection and 18 healthy controls and liver tissues from 34 patients were examined to assess the protein expression of NLRP3 and its activation marker CASP1 using immunohistochemical staining. The results showed that the median serum NLRP3 levels was significantly higher in HCV-infected patients compared with healthy controls (1040 pg/ml vs 695 pg/ml respectively, P < 0.001) and were positively correlated with hepatic NLRP3 and CASP1 expression (r = 0.749, P < 0.001 and r = 0.557, P = 0.001 respectively). The NLRP3 levels in serum and the liver significantly increased with worsening liver pathology and showed positive correlations with serum aminotransferases levels, HCV viremia, and albumin-bilirubin score (P < 0.05). The receiver operating characteristic curve analysis revealed a high diagnostic performance of serum NLRP3 in determining the extent of liver necroinflammation, fibrosis, and steatosis (area under the curve = 0.951, 0.971, and 0.917 respectively, P < 0.001). In conclusion, NLRP3 plays an important role in liver disease progression during HCV infection via CASP1 activation and might be a promising therapeutic target. Serum NLRP3 could be an additional biomarker for liver inflammation and fibrosis.


Assuntos
Hepacivirus , Hepatite C Crônica , Humanos , Caspase 1/metabolismo , Fibrose , Hepacivirus/metabolismo , Inflamassomos/metabolismo , Fígado/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR/metabolismo , Domínio Pirina
7.
J Virol ; 96(22): e0099722, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36314819

RESUMO

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral life cycle. This life cycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins methyltransferase-like 3 and 14 (METTL3 and METTL14) is regulated by Wilms' tumor 1-associating protein (WTAP). WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production was independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV life cycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic life cycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.


Assuntos
Proteínas de Ciclo Celular , Hepatite C , Metiltransferases , Fatores de Processamento de RNA , Humanos , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/genética , Hepatite C/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo
8.
J Phys Chem B ; 126(42): 8391-8403, 2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36255318

RESUMO

Hepatitis C virus (HCV) is the second viral agent that causes the majority of chronic hepatic infections worldwide, following Hepatitis B virus (HBV) infection. HCV infection comprises several steps, from the attachment to the receptors to the delivery of the viral genetic material and replication inside the cells. Tetraspanin CD81 is a key entry factor for HCV as it accompanies the virus during attachment and internalization through clathrin-mediated endocytosis. HCV-CD81 binding takes place through the viral glycoprotein E2. We performed full-atom molecular dynamics simulations reproducing the pH conditions that occur during the viral attachment to the hepatocytes (pH 7.4) and internalization (pH 6.2-4.6). We observed that changing the pH from 7.4 to 6.2 triggers a large conformational change in the binding orientation between E2core (E2core corresponds to residues 412-645 of the viral glycoprotein E2) and CD81LEL (CD81LEL corresponds to residues 112-204 of CD81) that occurs even more rapidly at low pH 4.6. This pH-induced switching mechanism has never been observed before and could allow the virus particles to sense the right moment during the maturation of the endosome to start fusion.


Assuntos
Hepacivirus , Hepatite C , Humanos , Tetraspanina 28/química , Tetraspanina 28/metabolismo , Hepacivirus/metabolismo , Proteínas do Envelope Viral/química , Clatrina/metabolismo
9.
Mol Microbiol ; 118(5): 570-587, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36203260

RESUMO

Hepatitis C virus (HCV) infection is one of the most common causes of liver cancer. HCV infection causes chronic disease followed by cirrhosis, which often leads to hepatocellular carcinoma (HCC). In this study, we investigated the roles of exosome-associated miRNAs in HCV-induced disease pathology. Small RNA sequencing was performed to identify miRNAs that are differentially regulated in exosomes isolated from patient sera at two different stages of HCV infection: cirrhosis and hepatocellular carcinoma. Among the differentially expressed miRNAs, miR-375 was found to be significantly upregulated in exosomes isolated from patients with cirrhosis and HCC. A similar upregulation was observed in intracellular and extracellular/exosomal levels of miR-375 in HCV-JFH1 infected Huh7.5 cells. The depletion of miR-375 in infected cells inhibited HCV-induced cell migration and proliferation, suggesting a supportive role for miR-375 in HCV pathogenesis. miR-375, secreted through exosomes derived from HCV-infected cells, could also be transferred to naïve Huh7.5 cells, resulting in an increase in cell proliferation and migration in the recipient cells. Furthermore, we identified Insulin growth factor binding protein 4 (IGFBP4), a gene involved in cell growth and malignancy, as a novel target of miR-375. Our results demonstrate the critical involvement of exosome-associated miR-375 in HCV-induced disease progression.


Assuntos
Carcinoma Hepatocelular , Exossomos , Hepatite C , Neoplasias Hepáticas , MicroRNAs , Humanos , Hepacivirus/genética , Hepacivirus/metabolismo , Exossomos/metabolismo , Exossomos/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Insulina/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Hepatite C/genética , Hepatite C/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proliferação de Células/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia
10.
Sci Rep ; 12(1): 16577, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36195643

RESUMO

Halophilic archaea is considered an promising natural source of many important metabolites. This study focused on one of the surface-active biomolecules named biosurfactants produced by haloarchaeon Natrialba sp. M6. The production trend was optimized and the product was partially purified and identified using GC-Mass spectrometry. Sequential optimization approaches, Plackett-Burman (PB) and Box-Behnken Designs (BBD) were applied to maximize the biosurfactants production from M6 strain by using 14 factors; pH, NaCl, agitation and glycerol; the most significant factors that influenced the biosurfactant production were used for Response Surface Methodology (RSM). The final optimal production conditions were agitation (150 rpm), glycerol (3%), NaCl (20.8%), pH (12) and cultivation temperature (37°C). GC-Mass spectrometry for the recovered extract revealed the presence of a diverse group of bipolar nature, hydrophobic hydrocarbon chain and charged function group. The majority of these compounds are fatty acids. Based on results of GC-MS, compositional analysis content and Zetasizer, it was proposed that the extracted biosurfactant produced by haloarchaeon Natrialba sp. M6 could be a cationic lipoprotein. The antiviral activity of such biosurfactant was investigated against hepatitis C (HCV) and herpes simplex (HSV1) viruses at its maximum safe doses (20 µg/mL and 8 µg/mL, respectively). Its mode of antiviral action was declared to be primarily via deactivating viral envelopes thus preventing viral entry. Moreover, this biosurfactant inhibited RNA polymerase- and DNA polymerase-mediated viral replication at IC50 of 2.28 and 4.39 µg/mL, respectively also. Molecular docking studies showed that surfactin resided well and was bound to the specified motif with low and accepted binding energies (ΔG = - 5.629, - 6.997 kcal/mol) respectively. Therefore, such biosurfactant could be presented as a natural safe and effective novel antiviral agent.


Assuntos
Hepatite C , Herpes Simples , Antivirais/farmacologia , DNA Polimerase Dirigida por DNA , Ácidos Graxos , Glicerol , Halobacteriaceae , Hepacivirus/metabolismo , Humanos , Simulação de Acoplamento Molecular , Cloreto de Sódio , Tensoativos/química
11.
Front Immunol ; 13: 953151, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36159876

RESUMO

Chronic hepatitis C virus (HCV) infection is a curable disease, but the absence of a vaccine remains a major problem in infection prevention. The lack of small animal models and limited access to human liver tissue impede the study of hepatic antiviral immunity and the development of new vaccine strategies. We recently developed an immune-competent mouse model using an HCV-related rodent hepacivirus which shares immunological features with human viral hepatitis. In this study, we used this new model to investigate the role of invariant natural killer T (iNKT) cells during hepacivirus infection in vivo. These cells are enriched in the liver, however their role in viral hepatitis is not well defined. Using high-dimensional flow cytometry and NKT cell deficient mice we analyzed a potential role of iNKT cells in mediating viral clearance, liver pathology or immune-regulation during hepacivirus infection. In addition, we identified new immune-dominant MHC class I restricted viral epitopes and analyzed the impact of iNKT cells on virus-specific CD8+ T cells. We found that rodent hepacivirus infection induced the activation of iNKT cell subsets with a mixed NKT1/NKT2 signature and significant production of type 2 cytokines (IL-4 and IL-13) during acute infection. While iNKT cells were dispensable for viral clearance, the lack of these cells caused higher levels of liver injury during infection. In addition, the absence of iNKT cells resulted in increased effector functions of hepatic antiviral T cells. In conclusion, our study reports a regulatory role of hepatic iNKT cells during hepacivirus infection in vivo. Specifically, our data suggest that iNKT cells skewed towards type 2 immunity limit liver injury during acute infection by mechanisms that include the regulation of effector functions of virus-specific T cells.


Assuntos
Hepatite C Crônica , Células T Matadoras Naturais , Animais , Antivirais/metabolismo , Citocinas/metabolismo , Epitopos/metabolismo , Hepacivirus/metabolismo , Humanos , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Camundongos
12.
J Biol Chem ; 298(11): 102486, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36108740

RESUMO

Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.


Assuntos
Hepatite C , Neoplasias Hepáticas , Humanos , Proteínas não Estruturais Virais/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Hepatite C/metabolismo , RNA Helicases/metabolismo
13.
FEBS Lett ; 596(19): 2525-2537, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35918185

RESUMO

The tumour suppressor p53 has been implicated in the host defence system against hepatitis C virus (HCV) infection, although the detailed mechanism remains unknown. Here, we found that p53 inhibits HCV replication by downregulating HCV Core protein levels in human hepatoma cells. For this effect, p53 potentiated the role of E6-associated protein (E6AP) as an E3 ligase to induce ubiquitination and proteasomal degradation of HCV Core. Specifically, p53 facilitated the binding of E6AP to HCV Core through direct interactions with the two proteins. In addition, E6AP failed to induce ubiquitination of HCV Core in the absence of p53, suggesting that p53 increases the E3 ligase activity of E6AP in a triple complex consisting of p53, E6AP and HCV Core.


Assuntos
Hepatite C , Neoplasias , Proteínas Oncogênicas Virais , Hepacivirus/metabolismo , Humanos , Proteínas Oncogênicas Virais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas do Core Viral/metabolismo , Replicação Viral
14.
Mol Cells ; 45(10): 702-717, 2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-35993162

RESUMO

Hepatitis C virus (HCV) infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV employs diverse strategies to evade host antiviral innate immune responses to mediate a persistent infection. In the present study, we show that nonstructural protein 5A (NS5A) interacts with an NF-κB inhibitor immunomodulatory kinase, IKKε, and subsequently downregulats beta interferon (IFN-ß) promoter activity. We further demonstrate that NS5A inhibits DDX3-mediated IKKε and interferon regulatory factor 3 (IRF3) phosphorylation. We also note that hyperphosphorylation of NS5A mediats protein interplay between NS5A and IKKε, thereby contributing to NS5A-mediated modulation of IFN-ß signaling. Lastly, NS5A inhibits IKKε-dependent p65 phosphorylation and NF-κB activation. Based on these findings, we propose NS5A as a novel regulator of IFN signaling events, specifically by inhibiting IKKε downstream signaling cascades through its interaction with IKKε. Taken together, these data suggest an additional mechanistic means by which HCV modulates host antiviral innate immune responses to promote persistent viral infection.


Assuntos
Hepatite C , Proteínas não Estruturais Virais , Antivirais , Hepacivirus/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , NF-kappa B/metabolismo , Proteínas não Estruturais Virais/metabolismo
15.
Pol J Pathol ; 73(2): 88-98, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35979755

RESUMO

The pathogenesis of hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) differs according to whether prior treatment with interferon (IFN) vs. direct-acting antiviral agents (DAAs) was administered. Cyclo- oxygenase-2 (COX-2), yes-associated protein 1 (YAP), and transcriptional co-activator with PDZ-binding motif (TAZ) play a crucial role in hepatocarcinogenesis. However, their roles in untreated or treated HCV-related HCC development have not been clarified. Therefore, we performed an immunohistochemical study and stained tissue from 83 HCV-related HCC cases using antibodies against COX-2, YAP, and TAZ and correlated their expression with the clinicopathological characteri stics and survival data. The cases were subdivided into 3 groups based on prior HCV treatment. In the 3 groups, COX-2 was significantly higher in HCC tissue compared with adjacent non-tumour liver tissue. However, the expression of YAP/TAZ was not significantly different between HCC and adjacent non-tumour tissue. We further grouped HCC cases into YAP+/TAZ+ and YAP-/TAZ- cases. In the YAP+/TAZ+ cases, COX-2 was significantly associated with tumour size, tumour multifocality, and late pathologic stage. No significant difference was observed in COX-2 and TAZ expression as a result of IFN or DAA treatment; however, YAP was significantly higher in IFN-treated HCC. Cyclo-oxygenase-2 overexpression may play a role in late HCC development, while YAP/TAZ could play an early role in HCC progression. Sustained expression of combined YAP/TAZ could mediate the poor prognostic role of COX-2.


Assuntos
Carcinoma Hepatocelular , Hepatite C Crônica , Hepatite C , Neoplasias Hepáticas , Humanos , Antivirais/uso terapêutico , Ciclo-Oxigenase 2/uso terapêutico , Hepacivirus/metabolismo , Hepatite C/complicações , Hepatite C/tratamento farmacológico , Hepatite C Crônica/complicações , Hepatite C Crônica/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Transativadores/metabolismo , Transativadores/uso terapêutico , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional
16.
Front Public Health ; 10: 911551, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35923969

RESUMO

Background: Increased interferon (IFN)-gamma inducible protein-10 (IP-10) level has been shown to be associated with sustained virologic responses (SVRs) to pegylated interferon-alpha 2a/ribavirin-based therapy in patients with chronic hepatitis C (CHC). We investigated the relationship between IP-10 and treatment response in patients with CHC treated with direct-acting antiviral agents (DAAs) therapy. Methods: We measured the dynamic changes of IP-10 in samples from 90 patients with CHC. The serum IP-10 levels, intrahepatic expressions of IP-10 mRNA, and protein were determined, respectively. For the in vitro experiments, the expression changes of IP-10 in hepatitis C virus (HCV)-replicating Huh-7 cells with or without non-structural protein 5A (NS5A) inhibitor were analyzed using real-time reverse transcription-polymerase chain reaction and Western blotting. Results: Patients with chronic hepatitis C had increased baseline IP-10 levels, intrahepatic IP-10 mRNA, and protein expression. After initiating DAAs therapy, serum IP-10 levels decreased gradually in patients who achieved cure, whereas in patients who failed the therapy, IP-10 levels did not change significantly or recovered from the initial decline. Multivariate logistic regression analysis confirmed that baseline IP-10 level ≤ 450 pg/ml and decline >30% at 12 weeks independently predicted the SVR in patients with CHC who received DAAs. In vitro, the expression of IP-10 mRNA and protein in HCV-replicating Huh-7 cells increased significantly. However, such activities were downregulated by NS5A inhibitor, followed by the reduction of HCV RNA levels and a decline in IP-10 levels. Conclusion: IP-10 interfered with HCV replication in hepatocytes and the dynamic decline in IP-10 levels during DAA treatment predicted the SVR in patients with CHC.


Assuntos
Hepatite C Crônica , Hepatite C , Antivirais/farmacologia , Antivirais/uso terapêutico , Quimiocina CXCL10/metabolismo , Quimiocina CXCL10/uso terapêutico , Quimioterapia Combinada , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C Crônica/tratamento farmacológico , Humanos , RNA Mensageiro/uso terapêutico
17.
Viruses ; 14(8)2022 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-36016316

RESUMO

Hepatitis C virus is the major cause of chronic liver diseases and the only cytoplasmic RNA virus known to be oncogenic in humans. The viral genome gives rise to ten mature proteins and to additional proteins, which are the products of alternative translation initiation mechanisms. A protein-known as ARFP (alternative reading frame protein) or Core+1 protein-is synthesized by an open reading frame overlapping the HCV Core coding region in the (+1) frame of genotype 1a. Almost 20 years after its discovery, we still know little of the biological role of the ARFP/Core+1 protein. Here, our differential proteomic analysis of stable hepatoma cell lines expressing the Core+1/Long isoform of HCV-1a relates the expression of the Core+1/Long isoform with the progression of the pathology of HCV liver disease to cancer.


Assuntos
Carcinoma Hepatocelular , Hepatite C , Hepacivirus/genética , Hepacivirus/metabolismo , Antígenos da Hepatite C , Humanos , Isoformas de Proteínas/metabolismo , Proteômica , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo
18.
Int J Mol Sci ; 23(14)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35887291

RESUMO

Transmembrane drug transport in hepatocytes is one of the major determinants of drug pharmacokinetics. In the present study, ABC transporters (P-gp, MRP1, MRP2, MRP3, MRP4, BCRP, and BSEP) and SLC transporters (MCT1, NTCP, OAT2, OATP1B1, OATP1B3, OATP2B1, OCT1, and OCT3) were quantified for protein abundance (LC-MS/MS) and mRNA levels (qRT-PCR) in hepatitis C virus (HCV)-infected liver samples from the Child-Pugh class A (n = 30), B (n = 21), and C (n = 7) patients. Protein levels of BSEP, MRP3, MCT1, OAT2, OATP1B3, and OCT3 were not significantly affected by HCV infection. P-gp, MRP1, BCRP, and OATP1B3 protein abundances were upregulated, whereas those of MRP2, MRP4, NTCP, OATP2B1, and OCT1 were downregulated in all HCV samples. The observed changes started to be seen in the Child-Pugh class A livers, i.e., upregulation of P-gp and MRP1 and downregulation of MRP2, MRP4, BCRP, and OATP1B3. In the case of NTCP, OATP2B1, and OCT1, a decrease in the protein levels was observed in the class B livers. In the class C livers, no other changes were noted than those in the class A and B patients. The results of the study demonstrate that drug transporter protein abundances are affected by the functional state of the liver in hepatitis C patients.


Assuntos
Hepatite C , Transportadores de Ânions Orgânicos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Cromatografia Líquida/métodos , Hepacivirus/metabolismo , Hepatite C/metabolismo , Humanos , Fígado/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Espectrometria de Massas em Tandem/métodos
19.
Elife ; 112022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35796426

RESUMO

E1 and E2 (E1E2), the fusion proteins of Hepatitis C Virus (HCV), are unlike that of any other virus yet described, and the detailed molecular mechanisms of HCV entry/fusion remain unknown. Hypervariable region-1 (HVR-1) of E2 is a putative intrinsically disordered protein tail. Here, we demonstrate that HVR-1 has an autoinhibitory function that suppresses the activity of E1E2 on free virions; this is dependent on its conformational entropy. Thus, HVR-1 is akin to a safety catch that prevents premature triggering of E1E2 activity. Crucially, this mechanism is turned off by host receptor interactions at the cell surface to allow entry. Mutations that reduce conformational entropy in HVR-1, or genetic deletion of HVR-1, turn off the safety catch to generate hyper-reactive HCV that exhibits enhanced virus entry but is thermally unstable and acutely sensitive to neutralising antibodies. Therefore, the HVR-1 safety catch controls the efficiency of virus entry and maintains resistance to neutralising antibodies. This discovery provides an explanation for the ability of HCV to persist in the face of continual immune assault and represents a novel regulatory mechanism that is likely to be found in other viral fusion machinery.


Assuntos
Hepacivirus , Hepatite C , Anticorpos Neutralizantes , Entropia , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus
20.
EMBO J ; 41(16): e110581, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35822879

RESUMO

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.


Assuntos
Hepacivirus , Hepatite C , Fator de Iniciação 2 em Eucariotos/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Ribossomos/metabolismo
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