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1.
Front Immunol ; 12: 654998, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34531848

RESUMO

HCV core protein is the first structural protein synthesized during hepatitis C virus (HCV) infection and replication. It is released from virus infected liver cells and mediates multiple functions to affect host cell response. The innate immune response is the first line of defense against viral infection. After HCV infection, Kupffer cells (KCs) which are liver macrophages play an important role in host innate immune response. Kupffer cells act as phagocytes and release different cytokines and chemokines to counter viral infection and regulate inflammation and fibrosis in liver. Earlier, we have demonstrated that HCV core protein interacts with gC1qR and activates MAPK, NF-κB and PI3K/AKT pathways in macrophages. In this study, we explored the effect of HCV core protein on CCL2 and CXCL10 expression in macrophages and the signaling pathways involved. Upon silencing of gC1qR, we observed a significant decrease expression of CCL2 and CXCL10 in macrophages in the presence of HCV core protein. Inhibiting NF-κB pathway, but not P38, JNK, ERK and AKT pathways greatly reduced the expression of CCL2 and CXCL10. Therefore, our results indicate that interaction of HCV core protein with gC1qR could induce CCL2 and CXCL10 secretion in macrophages via NF-κB signaling pathway. These findings may shed light on the understanding of how leukocytes migrate into the liver and exaggerate host-derived immune responses and may provide novel therapeutic targets in HCV chronic inflammation.


Assuntos
Quimiocina CCL2/imunologia , Quimiocina CXCL10/imunologia , Hepacivirus/imunologia , Macrófagos/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Proteínas do Core Viral/imunologia , Animais , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Expressão Gênica/imunologia , Hepacivirus/metabolismo , Hepacivirus/fisiologia , Hepatite C/imunologia , Hepatite C/metabolismo , Hepatite C/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos do Fígado/imunologia , Macrófagos do Fígado/metabolismo , Macrófagos do Fígado/virologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Células THP-1 , Proteínas do Core Viral/metabolismo
2.
Cells ; 10(9)2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34571900

RESUMO

Iron is crucial to the regulation of the host innate immune system and the outcome of many infections. Hepatitis C virus (HCV), one of the major viral human pathogens that depends on iron to complete its life cycle, is highly skilled in evading the immune system. This study presents the construction and validation of a physiologically relevant triple-cell co-culture model that was used to investigate the input of iron in HCV infection and the interplay between HCV, iron, and determinants of host innate immunity. We recorded the expression patterns of key proteins of iron homeostasis involved in iron import, export and storage and examined their relation to the iron regulatory hormone hepcidin in hepatocytes, enterocytes and macrophages in the presence and absence of HCV. We then assessed the transcriptional profiles of pro-inflammatory cytokines Interleukin-6 (IL-6) and interleukin-15 (IL-15) and anti-inflammatory interleukin-10 (IL-10) under normal or iron-depleted conditions and determined how these were affected by infection. Our data suggest the presence of a link between iron homeostasis and innate immunity unfolding among liver, intestine, and macrophages, which could participate in the deregulation of innate immune responses observed in early HCV infection. Coupled with iron-assisted enhanced viral propagation, such a mechanism may be important for the establishment of viral persistence and the ensuing chronic liver disease.


Assuntos
Enterócitos/patologia , Hepatite C/patologia , Hepatócitos/patologia , Homeostase , Imunidade Inata , Ferro/metabolismo , Macrófagos/patologia , Técnicas de Cocultura , Citocinas/metabolismo , Enterócitos/imunologia , Enterócitos/metabolismo , Enterócitos/virologia , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepatite C/imunologia , Hepatite C/metabolismo , Hepatite C/virologia , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia
3.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360721

RESUMO

Host lipid metabolism reprogramming is essential for hepatitis C virus (HCV) infection and progression to severe liver disease. Direct-acting antivirals (DAAs) achieve a sustained virological response (SVR) in most patients, but virus eradication does not always protect against hepatocellular carcinoma (HCC). Angiopoietin-like protein-3 (ANGPTL-3) and angiopoietin-like protein-4 (ANGPTL-4) regulate the clearance of plasma lipids by inhibiting cellular lipase activity and possess emerging roles in tumourigenesis. We used ELISA and RT-qPCR to investigate ANGPTL-3 and ANGPTL-4 expression in HCV patients with characterised fibrosis throughout the natural history of hepatitis C and in long-term HCV infection in vitro, before and after DAA treatment. ANGPTL-3 was decreased in patients with advanced fibrosis compared to other disease stages, while ANGPTL-4 was progressively increased from acute infection to cirrhosis and HCC, peaking at the advanced fibrosis stage. Only ANGPTL-3 mRNA was down-regulated during early infection in vitro, although both ANGPTLs were increased later. DAA treatment did not alter ANGPTL-3 levels in advanced fibrosis/cirrhosis and in HCV infection in vitro, in contrast to ANGPTL-4. The association between ANGPTLs and fibrosis in HCV infection was underlined by an inverse correlation between the levels of ANGPTLs and serum transforming growth factor- ß (TGF-ß). Collectively, we demonstrate the pivotal role of advanced fibrosis in defining the expression fate of ANGPTLs in HCV infection and after treatment and propose a role for ANGPTL-3 as a contributor to post-treatment deregulation of lipid metabolism that could predispose certain individuals to HCC development.


Assuntos
Proteína 4 Semelhante a Angiopoietina/biossíntese , Proteínas Semelhantes a Angiopoietina/biossíntese , Antivirais/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Hepacivirus/metabolismo , Hepatite C Crônica , Cirrose Hepática , Linhagem Celular Tumoral , Feminino , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/metabolismo , Humanos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Masculino
4.
J Med Virol ; 93(11): 6116-6123, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34375002

RESUMO

Virus invasion activates the host's innate immune response, inducing the production of numerous cytokines and interferons to eliminate pathogens. Except for viral DNA/RNA, viral proteins are also targets of pattern recognition receptors. Membrane-bound receptors such as Toll-like receptor (TLR)1, TLR2, TLR4, TLR6, and TLR10 relate to the recognition of viral proteins. Distinct TLRs perform both protective and detrimental roles for a specific virus. Here, we review viral proteins serving as pathogen-associated molecular patterns and their corresponding TLRs. These viruses are all enveloped, including respiratory syncytial virus, hepatitis C virus, measles virus, herpesvirus human immunodeficiency virus, and coronavirus, and can encode proteins to activate innate immunity in a TLR-dependent way. The TLR-viral protein relationship plays an important role in innate immunity activation. A detailed understanding of their pathways contributes to a novel direction for vaccine development.


Assuntos
Imunidade Inata , Padrões Moleculares Associados a Patógenos/metabolismo , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Proteínas Virais/metabolismo , Viroses/imunologia , Vírus/imunologia , Animais , HIV/imunologia , HIV/metabolismo , HIV/patogenicidade , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepacivirus/patogenicidade , Herpesviridae/imunologia , Herpesviridae/metabolismo , Herpesviridae/patogenicidade , Humanos , Vírus do Sarampo/imunologia , Vírus do Sarampo/metabolismo , Vírus do Sarampo/patogenicidade , Padrões Moleculares Associados a Patógenos/química , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/metabolismo , Vírus Sinciciais Respiratórios/patogenicidade , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Proteínas Virais/química , Viroses/virologia , Vírus/metabolismo , Vírus/patogenicidade
5.
Molecules ; 26(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202190

RESUMO

Background: The disease caused by hepatitis C virus (HCV) is asymptomatic, silent, and progressive liver disease. In HCV-infected patients the increase in serum HA is associated with the development of hepatic fibrosis and disease progression. Methods: HCV-RNA detection was performed in all serological samples of blood donors that tested positive using HCV Ultra ELISA. Determination of hyaluronan (HA) was performed in positive HCV samples using ELISA-like fluorometric method. The HA content was compared to HCV viral load, genotype of the virus, liver fibrosis as well as ALT and GGT liver biomarkers. Results: Persistently normal ALT (<40 U/L) and GGT (<50 U/L) serum levels were detected in 75% and 69% of the HCV-Infected blood donors, respectively. Based on ROC analysis, the HA value < 34.2 ng/mL is an optimal cut-off point to exclude HCV viremia (specificity = 91%, NPV = 99%). Applying HA value ≥34.2 ng/mL significant liver fibrosis (≥F2) can be estimated in 46% of the HCV-infected blood donors. HA serum level (≥34.2 ng/mL) associated with a high ALT level (>40 U/mL) can correctly identify HCV infection and probable liver fibrosis (sensitivity = 96% and specificity = 90%) in asymptomatic blood donors. Conclusions: A high level of HA (≥34.2 ng/mL) in association with ALT (≥40 U/L) in serum can provide a good clinical opportunity to detect HCV-infected asymptomatic persons that potentially require a liver biopsy confirmation and antiviral treatment to prevent the development of advanced liver fibrosis or cirrhosis.


Assuntos
Doadores de Sangue , Hepacivirus/metabolismo , Hepatite C/sangue , Hepatite C/diagnóstico , Ácido Hialurônico/sangue , Cirrose Hepática/sangue , Cirrose Hepática/diagnóstico , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Hepacivirus/genética , Hepatite C/genética , Humanos , Cirrose Hepática/genética , Masculino , Pessoa de Meia-Idade
6.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069902

RESUMO

Hepatocellular carcinoma (HCC) still remains a difficult to cure malignancy. In recent years, the focus has shifted to lipid metabolism for the treatment of HCC. Very little is known about hepatitis B virus (HBV) and C virus (HCV)-related hepatic lipid disturbances in non-malignant and cancer tissues. The present study showed that triacylglycerol and cholesterol concentrations were similar in tumor adjacent HBV and HCV liver, and were not induced in the HCC tissues. Higher levels of free cholesterol, polyunsaturated phospholipids and diacylglycerol species were noted in non-tumorous HBV compared to HCV liver. Moreover, polyunsaturated phospholipids and diacylglycerols, and ceramides declined in tumors of HBV infected patients. All of these lipids remained unchanged in HCV-related HCC. In HCV tumors, polyunsaturated phosphatidylinositol levels were even induced. There were no associations of these lipid classes in non-tumor tissues with hepatic inflammation and fibrosis scores. Moreover, these lipids did not correlate with tumor grade or T-stage in HCC tissues. Lipid reprogramming of the three analysed HBV/HCV related tumors mostly resembled HBV-HCC. Indeed, lipid composition of non-tumorous HCV tissue, HCV tumors, HBV tumors and HBV/HCV tumors was highly similar. The tumor suppressor protein p53 regulates lipid metabolism. The p53 and p53S392 protein levels were induced in the tumors of HBV, HCV and double infected patients, and this was significant in HBV infection. Negative correlation of tumor p53 protein with free cholesterol indicates a role of p53 in cholesterol metabolism. In summary, the current study suggests that therapeutic strategies to target lipid metabolism in chronic viral hepatitis and associated cancers have to consider disease etiology.


Assuntos
Carcinoma Hepatocelular/metabolismo , Colesterol/metabolismo , Fígado/metabolismo , Adulto , Idoso , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/genética , Colesterol/fisiologia , Feminino , Alemanha/epidemiologia , Hepacivirus/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/metabolismo , Hepatite C/virologia , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade
7.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925399

RESUMO

Recent evidence suggests that fibrotic liver injury in patients with chronic hepatitis C correlates with cellular senescence in damaged liver tissue. However, it is still unclear how senescence can affect replication of the hepatitis C virus (HCV). In this work, we report that an inhibitor of cyclin-dependent kinases 4/6, palbociclib, not only induced in hepatoma cells a pre-senescent cellular phenotype, including G1 arrest in the cell cycle, but also accelerated viral replicon multiplication. Importantly, suppression of HCV replication by direct acting antivirals (DAAs) was barely affected by pre-senescence induction, and vice versa, the antiviral activities of host-targeting agents (HTAs), such as inhibitors of human histone deacetylases (HDACi), produced a wide range of reactions-from a dramatic reduction to a noticeable increase. It is very likely that under conditions of the G1 arrest in the cell cycle, HDACi exhibit their actual antiviral potency, since their inherent anticancer activity that complicates the interpretation of test results is minimized.


Assuntos
Senescência Celular/fisiologia , Hepacivirus/metabolismo , Replicação Viral/fisiologia , Antivirais/farmacologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Genótipo , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Humanos , Fígado/patologia , Fenótipo , Piperazinas/farmacologia , Piridinas/farmacologia , Fator de Crescimento Transformador beta1/metabolismo
8.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649237

RESUMO

Hepatitis C virus (HCV) infections are associated with the risk of progression to fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV RNA genome is translated by an internal ribosome entry site (IRES)-dependent mechanism. The structure and function of the HCV IRES have been investigated by both biological and biophysical criteria. Recently, the role of N6-methyladenosine (m6A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m6A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m6A modification of the HCV genome in the IRES-dependent translation function by mutating m6A consensus motifs (DRACH) within the IRES element in stem-loop III and IV regions and studied their effect on translation initiation. There are several DRACH motifs within the IRES element. Of these, the DRACH motif at nucleotide (nt) 329-333, located about 7 nt upstream of initiator AUG (iAUG) codon, regulates IRES-mediated translation initiation. Mutational analysis showed that m6A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m6A reader protein YTHDC2, containing the RNA helicase domain, recognizes m6A-methylated adenosine at nt 331 and, in concert with the cellular La antigen, supports HCV IRES-dependent translation. The RNA helicase dead YTHDC2 (E332Q) mutant failed to stimulate HCV translation initiation. This report highlights the functional roles of m6A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.


Assuntos
Adenosina/análogos & derivados , Genoma Viral , Hepacivirus/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Hepacivirus/genética , Humanos , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Helicases/genética , RNA Viral/genética
9.
Biomed Pharmacother ; 136: 111239, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33454599

RESUMO

Hepatitis C is an inflammatory liver disease caused by the single-stranded RNA (ssRNA) hepatitis C virus (HCV). The genetic diversity of the virus and quasispecies produced during replication have resulted in viral resistance to direct-acting antivirals (DAAs) as well as impediments in vaccine development. The recent adaptation of CRISPR-Cas as an alternative antiviral approach has demonstrated degradation of viral nucleic acids in eukaryotes. In particular, the CRISPR-effector Cas13 enzyme has been shown to target ssRNA viruses effectively. In this work, we have employed Cas13a to knockdown HCV in mammalian cells. Using a computational screen, we identified several potential Cas13a target sites within highly conserved regions of the HCV internal ribosomal entry site (IRES). Our results demonstrate significant inhibition of HCV replication as well as translation in huh-7.5 cells with minimal effects on cell viability. These findings were validated using a multi-modality approach involving qRT-PCR, luciferase assay, and MTT cell viability assay. In conclusion, the CRISPR-Cas13a system efficiently targets HCV in vitro, suggesting its potential as a programmable therapeutic antiviral strategy.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Marcação de Genes , Hepacivirus/genética , Hepatite C/terapia , Sítios Internos de Entrada Ribossomal , RNA Viral/genética , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular Tumoral , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/metabolismo , Hepatite C/genética , Hepatite C/virologia , Humanos , Estabilidade de RNA , RNA Viral/metabolismo , Replicação Viral/efeitos dos fármacos
10.
Ann Hematol ; 100(2): 321-336, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33079221

RESUMO

To ensure the safety of high-dose chemotherapy and autologous stem cell transplantation (HDC/ASCT), evidence-based recommendations on infectious complications after HDC/ASCT are given. This guideline not only focuses on patients with haematological malignancies but also addresses the specifics of HDC/ASCT patients with solid tumours or autoimmune disorders. In addition to HBV and HCV, HEV screening is nowadays mandatory prior to ASCT. For patients with HBs antigen and/or anti-HBc antibody positivity, HBV nucleic acid testing is strongly recommended for 6 months after HDC/ASCT or for the duration of a respective maintenance therapy. Prevention of VZV reactivation by vaccination is strongly recommended. Cotrimoxazole for the prevention of Pneumocystis jirovecii is supported. Invasive fungal diseases are less frequent after HDC/ASCT, therefore, primary systemic antifungal prophylaxis is not recommended. Data do not support a benefit of protective room ventilation e.g. HEPA filtration. Thus, AGIHO only supports this technique with marginal strength. Fluoroquinolone prophylaxis is recommended to prevent bacterial infections, although a survival advantage has not been demonstrated.


Assuntos
Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas , Hepacivirus/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/prevenção & controle , Hepatite C/prevenção & controle , Pneumocystis carinii/metabolismo , Pneumonia por Pneumocystis/prevenção & controle , Alemanha , Hematologia , Hepatite B/sangue , Anticorpos Anti-Hepatite B/sangue , Hepatite C/sangue , Humanos , Oncologia , Pneumonia por Pneumocystis/sangue , Guias de Prática Clínica como Assunto , RNA Viral/sangue , Sociedades Médicas , Transplante Autólogo , Combinação Trimetoprima e Sulfametoxazol/uso terapêutico
11.
Virus Res ; 292: 198251, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33259871

RESUMO

The resistance of hepatitis C virus (HCV) to direct-acting antiviral agents, used in chronic hepatitis C treatment, consists of a natural process resulting from resistance-associated substitutions (RASs) at specific amino acid regions. To identify and establish the natural prevalence of RASs in the NS3 gene in patients with chronic hepatitis C in the state of Pará, northern Brazil. Molecular analysis was performed on a total of 35 patients infected with HCV genotype 1, who were treatment-naive to protease inhibitors. HCV RNA was extracted from plasma and the NS3 region was amplified and submitted to DNA sequencing (Sanger). The general natural prevalence of RASs in the NS3 gene was 37.5 % (Y56F and S122T). The substitutions Y56F (34.3 %), S122T (3.1 %), V132I (15.6 %) and V170I (9.3 %) were identified. Y56F and S122T provide resistance to the protease inhibitors grazoprevir and simeprevir, respectively. All amino acid substitutions in the NS3 gene, including RASs, identified in patients from the state of Pará were present in other Brazilian studies. The natural presence of RASs in this study reflects the elevated genetic variability of HCV.


Assuntos
Substituição de Aminoácidos , Hepacivirus/genética , Hepatite C Crônica/virologia , Proteínas não Estruturais Virais/genética , Adulto , Idoso , Antivirais/farmacologia , Brasil , Farmacorresistência Viral , Feminino , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Estudos Prospectivos , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/metabolismo
12.
J Microbiol ; 59(1): 101-109, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33355889

RESUMO

Hepatitis C virus (HCV) life cycle is highly dependent on cellular proteins for viral propagation. In order to identify the cellular factors involved in HCV propagation, we previously performed a protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, adenosylhomocysteinase like 1 (AHCYL1) was among 90 proteins identified as NS5A interactors. Of these candidates, AHCYL1 was selected for further study. In the present study, we verified the physical interaction between NS5A and AHCYL1 by both in vitro pulldown and coimmunoprecipitation assays. Furthermore, HCV NS5A interacted with endogenous AHCYL1 in Jc1-infected cells. Both NS5A and AHCYL1 were colocalized in the cytoplasmic region in HCV-replicating cells. siRNAmediated knockdown of AHCYL1 abrogated HCV propagation. Exogenous expression of the siRNA-resistant AHCYL1 mutant, but not of the wild-type AHCYL1, restored HCV protein expression levels, indicating that AHCYL1 was required specifically for HCV propagation. Importantly, AHCYL1 was involved in the HCV internal ribosome entry site-mediated translation step of the HCV life cycle. Finally, we demonstrated that the proteasomal degradation pathway of AHCYL1 was modulated by persistent HCV infection. Collectively, these data suggest that HCV may modulate the AHCYL1 protein to promote viral propagation.


Assuntos
Hepacivirus/metabolismo , Hepatite C/enzimologia , Proteínas não Estruturais Virais/metabolismo , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepatite C/genética , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Proteínas não Estruturais Virais/genética
13.
Biochim Biophys Acta Biomembr ; 1863(3): 183537, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33383025

RESUMO

Many viruses destabilize cellular membranous compartments to form their replication complexes, but the mechanism(s) underlying membrane perturbation remains unknown. Expression in eukaryotic cells of NS4B, a protein of the hepatitis C virus (HCV), alters membranous complexes and induces structures similar to the so-called membranous web that appears crucial to the formation of the HCV replication complex. As over-expression of the protein is lethal to both prokaryotic and eukaryotic cells, NS4B was produced in large quantities in a "cell-free" system in the presence of detergent, after which it was inserted into lipid membranes. X-ray diffraction revealed that NS4B modifies the phase diagram of synthetic lipid aqueous phases considerably, perturbing the transition temperature and cooperativity. Cryo-electron microscopy demonstrated that NS4B introduces significant disorder in the synthetic membrane as well as discontinuities that could be interpreted as due to the formation of pores and membrane merging events. C- and N-terminal fragments of NS4B are both able to destabilize liposomes. While most NS4B amphipathic peptides perforate membranes, one NS4B peptide induces membrane fusion. Cryo-electron microscopy reveals a particular structure that can be interpreted as arising from hemi-fusion-like events. Amphipathic domains are present in many proteins, and if exposed to the aqueous cytoplasmic medium are sufficient to destabilize membranes in order to form viral replication complexes. These domains have important functions in the viral replication cycle, and thus represent potential targets for the development of anti-viral molecules.


Assuntos
Hepacivirus/química , Membranas Artificiais , Peptídeos/química , Proteínas não Estruturais Virais/química , Hepacivirus/metabolismo , Peptídeos/metabolismo , Proteínas não Estruturais Virais/metabolismo
14.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33328315

RESUMO

Hepatitis C virus (HCV) infection causes liver pathologies, including hepatocellular carcinoma (HCC). Homeobox (HOX) gene products regulate embryonic development and are associated with tumorigenesis, although the regulation of HOX genes by HCV infection has not been clarified in detail. We examined the effect of HCV infection on HOX gene expression. In this study, HCV infection induced more than half of the HOX genes and reduced the level of histone H2A monoubiquitination on lysine 119 (K119) (H2Aub), which represses HOX gene promoter activity. HCV infection also promoted proteasome-dependent degradation of RNF2, which is an E3 ligase mediating H2A monoubiquitination as a component of polycomb repressive complex 1. Since full-genomic replicon cells but not subgenomic replicon cells exhibited reduced RNF2 and H2Aub levels and induction of HOX genes, we focused on the core protein. Expression of the core protein reduced the amounts of RNF2 and H2Aub and induced HOX genes. Treatment with LY-411575, which can reduce HCV core protein expression via signal peptide peptidase (SPP) inhibition without affecting other viral proteins, dose-dependently restored the amounts of RNF2 and H2Aub in HCV-infected cells and impaired the induction of HOX genes and production of viral particles but not viral replication. The chromatin immunoprecipitation assay results also indicated infection- and proteasome-dependent reductions in H2Aub located in HOX gene promoters. These results suggest that HCV infection or core protein induces HOX genes by impairing histone H2A monoubiquitination via a reduction in the RNF2 level.IMPORTANCE Recently sustained virologic response can be achieved by direct-acting antiviral (DAA) therapy in most hepatitis C patients. Unfortunately, DAA therapy does not completely eliminate a risk of hepatocellular carcinoma (HCC). Several epigenetic factors, including histone modifications, are well known to contribute to hepatitis C virus (HCV)-associated HCC. However, the regulation of histone modifications by HCV infection has not been clarified in detail. In this study, our data suggest that HCV infection or HCV core protein expression impairs monoubiquitination of histone H2A K119 in the homeobox (HOX) gene promoter via destabilization of RNF2 and then induces HOX genes. Several lines of evidence suggest that the expression of several HOX genes is dysregulated in certain types of tumors. These findings reveal a novel mechanism of HCV-related histone modification and may provide information about new targets for diagnosis and prevention of HCC occurrence.


Assuntos
Genes Homeobox/genética , Hepacivirus/fisiologia , Histonas/metabolismo , Ubiquitinação/fisiologia , Linhagem Celular , Regulação da Expressão Gênica , Hepacivirus/metabolismo , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/virologia , Código das Histonas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas do Core Viral/metabolismo
15.
PLoS One ; 15(12): e0244112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382729

RESUMO

OBJECTIVES: Mucosal-associated invariant T (MAIT) cells have been shown to contribute in the pathogenesis of various liver diseases, including chronic hepatitis C virus (HCV) infection. This study was aimed at investigating the frequency, phenotype, and function of circulating MAIT cells, as well as their alterations after successful direct-acting antivirals (DAAs) in HCV-infected patients with or without HIV infection. METHODS: A total 85 patients (51 HCV-monoinfection and 34 HCV/HIV-coinfection), who received elbasvir/grazoprevir from a clinical trial and 20 healthy controls were included. MAIT cells in blood were characterized using flow cytometry at baseline and 24 weeks post-treatment. RESULTS: HCV-monoinfected and HCV/HIV-coinfected patients achieved similar sustained virological response rates (SVR24, 94.1% vs. 97.1%). Circulating MAIT cells in the monoinfection and coinfection groups were presented at low frequencies in comparison with healthy controls (median, 1.1% vs. 1.1% vs. 2.4%, P<0.001) and exhibited features of chronic activation and impaired functional capacity. A negative correlation between circulating MAIT cell frequency and liver stiffness assessed by magnetic resonance elastography was observed. Compared with baseline, increased in circulating MAIT cells after successful DAA therapy was mainly detected in HCV-monoinfected patients compared with HCV/HIV-coinfected individuals. Moreover, MAIT cell restoration was predominantly observed among patients with significant fibrosis to cirrhosis (F2-F4). CONCLUSIONS: These data indicated that dysregulation of MAIT cells might play a role in the progression of chronic HCV infection. Partial restoration of MAIT cell frequency and function was observed after successful DAA therapy, particularly in HCV-monoinfected patients.


Assuntos
Antivirais/administração & dosagem , Benzofuranos/administração & dosagem , Hepacivirus/imunologia , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Imidazóis/administração & dosagem , Células T Invariantes Associadas à Mucosa/imunologia , Quinoxalinas/administração & dosagem , Adulto , Combinação de Medicamentos , Feminino , Hepacivirus/metabolismo , Hepatite C Crônica/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Células T Invariantes Associadas à Mucosa/metabolismo
16.
Int J Mol Sci ; 21(24)2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33333737

RESUMO

Protein kinases (PKs) are enzymes that catalyze the transfer of the terminal phosphate group from ATP to a protein acceptor, mainly to serine, threonine, and tyrosine residues. PK catalyzed phosphorylation is critical to the regulation of cellular signaling pathways that affect crucial cell processes, such as growth, differentiation, and metabolism. PKs represent attractive targets for drugs against a wide spectrum of diseases, including viral infections. Two different approaches are being applied in the search for antivirals: compounds directed against viral targets (direct-acting antivirals, DAAs), or against cellular components essential for the viral life cycle (host-directed antivirals, HDAs). One of the main drawbacks of DAAs is the rapid emergence of drug-resistant viruses. In contrast, HDAs present a higher barrier to resistance development. This work reviews the use of chemicals that target cellular PKs as HDAs against virus of the Flaviviridae family (Flavivirus and Hepacivirus), thus being potentially valuable therapeutic targets in the control of these pathogens.


Assuntos
Infecções por Flaviviridae/tratamento farmacológico , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Antivirais/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caseína Quinase I/antagonistas & inibidores , Caseína Quinase I/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Flaviviridae/efeitos dos fármacos , Flaviviridae/enzimologia , Infecções por Flaviviridae/enzimologia , Hepacivirus/enzimologia , Hepacivirus/metabolismo , Hepatite C Crônica/metabolismo , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo
17.
Elife ; 92020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33169665

RESUMO

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.


Assuntos
Antivirais/farmacologia , Hepacivirus/metabolismo , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/química , Biomarcadores , Linhagem Celular , Cães , Descoberta de Drogas , Genótipo , Hepacivirus/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais/metabolismo
18.
Theranostics ; 10(26): 12223-12240, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33204339

RESUMO

Rationale: Many viral infections are known to activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway. However, the role of p38 activation in viral infection and the underlying mechanism remain unclear. The role of virus-hijacked p38 MAPK activation in viral infection was investigated in this study. Methods: The correlation of hepatitis C virus (HCV) infection and p38 activation was studied in patient tissues and primary human hepatocytes (PHHs) by immunohistochemistry and western blotting. Coimmunoprecipitation, GST pulldown and confocal microscopy were used to investigate the interaction of p38α and the HCV core protein. In vitro kinase assays and mass spectrometry were used to analyze the phosphorylation of the HCV core protein. Plaque assays, quantitative real time PCR (qRT-PCR), western blotting, siRNA and CRISPR/Cas9 were used to determine the effect of p38 activation on viral replication. Results: HCV infection was associated with p38 activation in clinical samples. HCV infection increased p38 phosphorylation by triggering the interaction of p38α and TGF-ß activated kinase 1 (MAP3K7) binding protein 1 (TAB1). TAB1-mediated p38α activation facilitated HCV replication, and pharmaceutical inhibition of p38α activation by SB203580 suppressed HCV infection at the viral assembly step. Activated p38α interacted with the N-terminal region of the HCV core protein and subsequently phosphorylated the HCV core protein, which promoted HCV core protein oligomerization, an essential step for viral assembly. As expected, SB203580 or the HCV core protein N-terminal peptide (CN-peptide) disrupted the p38α-HCV core protein interaction, efficiently impaired HCV assembly and impeded normal HCV replication in both cultured cells and primary human hepatocytes. Similarly, severe fever with thrombocytopenia syndrome virus (SFTSV), herpes simplex virus type 1 (HSV-1) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection also activated p38 MAPK. Most importantly, pharmacological blockage of p38 activation by SB203580 effectively inhibited SFTSV, HSV-1 and SARS-CoV-2. Conclusion: Our study shows that virus-hijacked p38 activation is a key event for viral replication and that pharmacological blockage of p38 activation is an antiviral strategy.


Assuntos
COVID-19/metabolismo , Hepacivirus/metabolismo , Hepatite C/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , COVID-19/virologia , Chlorocebus aethiops , Ativação Enzimática , Células HEK293 , Hepatite C/patologia , Hepatite C/virologia , Hepatócitos/metabolismo , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação , Piridinas/farmacologia , Células Vero , Proteínas do Core Viral/metabolismo , Replicação Viral/efeitos dos fármacos
19.
Channels (Austin) ; 14(1): 403-412, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33092458

RESUMO

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted an urgent need to identify effective medicines for the prevention and treatment of the disease. A comparative analysis between SARS-CoV-2 and Hepatitis C Virus (HCV) can expand the available knowledge regarding the virology and potential drug targets against these viruses. Interestingly, comparing HCV with SARS-CoV-2 reveals major similarities between them, ranging from the ion channels that are utilized, to the symptoms that are exhibited by patients. Via this comparative analysis, and from what is known about HCV, the most promising treatments for COVID-19 can focus on the reduction of viral load, treatment of pulmonary system damages, and reduction of inflammation. In particular, the drugs that show most potential in this regard include ritonavir, a combination of peg-IFN, and lumacaftor-ivacaftor. This review anaylses SARS-CoV-2 from the perspective of the role of ion homeostasis and channels in viral pathomechanism. We also highlight other novel treatment approaches that can be used for both treatment and prevention of COVID-19. The relevance of this review is to offer high-quality evidence that can be used as the basis for the identification of potential solutions to the COVID-19 pandemic.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Hepacivirus/metabolismo , Canais Iônicos/metabolismo , Pneumonia Viral/metabolismo , Animais , Betacoronavirus/patogenicidade , COVID-19 , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Hepacivirus/patogenicidade , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2
20.
Int J Mol Sci ; 21(21)2020 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-33114346

RESUMO

Hijacking and manipulation of host cell biosynthetic pathways by human enveloped viruses are essential for the viral lifecycle. Flaviviridae members, including hepatitis C, dengue and Zika viruses, extensively manipulate host lipid metabolism, underlining the importance of lipid droplets (LDs) in viral infection. LDs are dynamic cytoplasmic organelles that can act as sequestration platforms for a unique subset of host and viral proteins. Transient recruitment and mobilization of proteins to LDs during viral infection impacts host-cell biological properties, LD functionality and canonical protein functions. Notably, recent studies identified LDs in the nucleus and also identified that LDs are transported extracellularly via an autophagy-mediated mechanism, indicating a novel role for autophagy in Flaviviridae infections. These developments underline an unsuspected diversity and localization of LDs and potential moonlighting functions of LD-associated proteins during infection. This review summarizes recent breakthroughs concerning the LD hijacking activities of hepatitis C, dengue and Zika viruses and potential roles of cytoplasmic, nuclear and extracellular LD-associated viral proteins during infection.


Assuntos
Flaviviridae/patogenicidade , Gotículas Lipídicas/metabolismo , Proteínas Virais/metabolismo , Animais , Autofagia , Núcleo Celular/metabolismo , Vírus da Dengue/metabolismo , Vírus da Dengue/patogenicidade , Espaço Extracelular/metabolismo , Flaviviridae/metabolismo , Hepacivirus/metabolismo , Hepacivirus/patogenicidade , Humanos , Gotículas Lipídicas/virologia , Zika virus/metabolismo , Zika virus/patogenicidade
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