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1.
Biomed Pharmacother ; 170: 116077, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38154274

RESUMO

Hepatitis D virus (HDV), which co-infects or superinfects patients with hepatitis B virus, is estimated to affect 74 million people worldwide. Chronic hepatitis D is the most severe form of viral hepatitis and can result in liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC). Currently, there are no efficient HDV-specific drugs. Therefore, there is an urgent need for novel HDV therapies that can achieve a functional cure or even eliminate the viral infection. In the HDV life cycle, agents targeting the entry step of HDV infection preemptively reduce the intrahepatic viral RNA. Human sodium taurocholate co-transporting polypeptide (hNTCP), a transporter of bile acids on the plasma membrane of hepatocytes, is an essential entry receptor of HDV and is a promising molecular target against HDV infection. Here, we investigated the effect of ergosterol peroxide (EP) on HDV infection in vitro and in vivo. EP inhibited HDV infection of hNTCP-expressing dHuS-E/2 hepatocytes by interrupting the early fusion/endocytosis step of HDV entry. Furthermore, molecular modeling suggested that EP hinders LHBsAg binding to hNTCP by blocking access to S267 and V263. In addition, we generated hNTCP-expressing transgenic (Tg) C57BL/6 mice using the Cre/loxP system for in vivo study. EP reduced the liver HDV RNA level of HDV-challenged hNTCP-Cre Tg mice. Intriguingly, EP downregulated the mRNA level of liver IFN-γ. We demonstrate that EP is a bona fide HDV entry inhibitor that acts on hNTCP and has the potential for use in HDV therapies.


Assuntos
Carcinoma Hepatocelular , Hepatite D , Neoplasias Hepáticas , Simportadores , Camundongos , Animais , Humanos , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Camundongos Endogâmicos C57BL , Hepatite D/tratamento farmacológico , Hepatite D/patologia , Vírus da Hepatite B/fisiologia , Hepatócitos , Camundongos Transgênicos , Simportadores/metabolismo
2.
Vaccine ; 41(21): 3337-3346, 2023 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-37085450

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combination with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.


Assuntos
Infecções por Coronavirus , Coronavírus da Síndrome Respiratória do Oriente Médio , Vacinas Virais , Animais , Camundongos , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , Dipeptidil Peptidase 4 , Imunidade Celular , Camundongos Transgênicos , Adjuvantes Imunológicos , Proteínas Recombinantes , Vacinas de Subunidades Antigênicas , Glicoproteína da Espícula de Coronavírus
3.
Virol J ; 19(1): 163, 2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253859

RESUMO

BACKGROUND: Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is a small, defective RNA virus strongly associated with the most severe form of hepatitis and progressive chronic liver disease and cirrhosis. Chronic hepatitis D, resulting from HBV/HDV coinfection, is considered to be the most severe form of viral hepatitis and affects 12-20 million people worldwide. Involved in the endocytosis and exocytosis of cellular and viral proteins, clathrin contributes to the pathogenesis and morphogenesis of HDV. Previously, we demonstrated that HDV-I and -II large hepatitis delta antigens (HDAg-L) possess a putative clathrin box that interacts with clathrin heavy chain (CHC) and supports HDV assembly. METHODS: Virus assembly and vesicular trafficking of HDV virus-like particles (VLPs) were evaluated in Huh7 cells expressing HDV-I, -II and -III HDAg-L and hepatitis B surface antigen (HBsAg). To elucidate the interaction motif between HDAg-L and CHC, site-directed mutagenesis was performed to introduce mutations into HDAg-L and CHC and analyzed using coimmunoprecipitation or pull-down assays. RESULTS: Comparable to HDV-I virus-like particles (VLPs), HDV-III VLPs were produced at a similar level and secreted into the medium via clathrin-mediated post-Golgi vesicular trafficking. Mutation at F27 or E33 of CHC abolished the binding of CHC to the C-terminus of HDV-III HDAg-L. Mutation at W207 of HDV-III HDAg-L inhibited its association with CHC and interfered with HDV-III VLP formation. We elucidated mechanism of the binding of HDV-III HDAg-L to CHC and confirmed the pivotal role of clathrin binding in the assembly of genotype III HDV. CONCLUSIONS: A novel W box which was identified at the C terminus of HDV-III HDAg-L is known to differ from the conventional clathrin box but also interacts with CHC. The novel W box of HDAg-L constitutes a new molecular target for anti-HDV-III therapeutics.


Assuntos
Antígenos de Superfície da Hepatite B , Vírus Delta da Hepatite , Clatrina/metabolismo , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Genótipo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/química , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/metabolismo , Humanos , RNA Viral/metabolismo , Proteínas Virais/genética , Replicação Viral
4.
Biochem J ; 478(12): 2321-2337, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34076705

RESUMO

LMBD1 was previously demonstrated to regulate the endocytosis of insulin receptor on the cell surface and to mediate the export of cobalamin from the lysosomes to the cytosol, but little is known about its function in mitosis. In this study, interactome analysis data indicate that LMBD1 is involved in cytoskeleton regulation. Both immunoprecipitation and GST pulldown assays demonstrated the association of LMBD1 with tubulin. Immunofluorescence staining also showed the colocalization of LMBD1 with microtubule in both interphase and mitotic cells. LMBD1 specifically accelerates microtubule assembly dynamics in vitro and antagonizes the microtubule-disruptive effect of vinblastine. In addition, LMBRD1-knockdown impairs mitotic spindle formation, inhibits tubulin polymerization, and diminishes the mitosis-associated tubulin acetylation. The reduced acetylation can be reversed by ectopic expression of LMBD1 protein. These results suggest that LMBD1 protein stabilizes microtubule intermediates. Furthermore, embryonic fibroblasts derived from Lmbrd1 heterozygous knockout mice showed abnormality in microtubule formation, mitosis, and cell growth. Taken together, LMBD1 plays a pivotal role in regulating microtubule assembly that is essential for the process of cell mitosis.


Assuntos
Citoesqueleto/fisiologia , Microtúbulos/fisiologia , Mitose , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Tubulina (Proteína)/química , Animais , Ciclo Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte Nucleocitoplasmático/genética , Domínios e Motivos de Interação entre Proteínas , Fuso Acromático/fisiologia
6.
J Virol ; 94(13)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32295922

RESUMO

Type I and type III interferons (IFNs) are the frontline of antiviral defense mechanisms that trigger hundreds of downstream antiviral genes. In this study, we observed that MERS-CoV nucleocapsid (N) protein suppresses type I and type III IFN gene expression. The N protein suppresses Sendai virus-induced IFN-ß and IFN-λ1 by reducing their promoter activity and mRNA levels, as well as downstream IFN-stimulated genes (ISGs). Retinoic acid-inducible gene I (RIG-I) is known to recognize viral RNA and induce IFN expression through tripartite motif-containing protein 25 (TRIM25)-mediated ubiquitination of RIG-I caspase activation and recruitment domains (CARDs). We discovered that MERS-CoV N protein suppresses RIG-I-CARD-induced, but not MDA5-CARD-induced, IFN-ß and IFN-λ1 promoter activity. By interacting with TRIM25, N protein impedes RIG-I ubiquitination and activation and inhibits the phosphorylation of transcription factors IFN-regulatory factor 3 (IRF3) and NF-κB that are known to be important for IFN gene activation. By employing a recombinant Sindbis virus-EGFP replication system, we showed that viral N protein downregulated the production of not only IFN mRNA but also bioactive IFN proteins. Taken together, MERS-CoV N protein functions as an IFN antagonist. It suppresses RIG-I-induced type I and type III IFN production by interfering with TRIM25-mediated RIG-I ubiquitination. Our study sheds light on the pathogenic mechanism of how MERS-CoV causes disease.IMPORTANCE MERS-CoV causes death of about 35% of patients. Published studies showed that some coronaviruses are capable of suppressing interferon (IFN) expression in the early phase of infection and MERS-CoV proteins can modulate host immune response. In this study, we demonstrated that MERS-CoV nucleocapsid (N) protein suppresses the production of both type I and type III IFNs via sequestering TRIM25, an E3 ubiquitin ligase that is essential for activating the RIG-I signaling pathway. Ectopic expression of TRIM25 rescues the suppressive effect of the N protein. In addition, the C-terminal domain of the viral N protein plays a pivotal role in the suppression of IFN-ß promoter activity. Our findings reveal how MERS-CoV evades innate immunity and provide insights into the interplay between host immune response and viral pathogenicity.


Assuntos
Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Proteína DEAD-box 58/metabolismo , Interferon Tipo I/biossíntese , Interferons/biossíntese , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Proteínas do Nucleocapsídeo/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linhagem Celular , Infecções por Coronavirus/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/genética , Interferons/genética , Regiões Promotoras Genéticas , Ligação Proteica , Receptores Imunológicos , Fatores de Transcrição , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Interferon lambda
7.
J Virol ; 93(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31462559

RESUMO

Hepatitis C virus (HCV) NS3 protein possesses protease and helicase activities and is considered an oncoprotein in virus-derived hepatocellular carcinoma. The NS3-associated oncogenesis has been studied but not fully understood. In this study, we have identified novel interactions of the NS3 protein with DNA repair factors, Werner syndrome protein (WRN) and Ku70, in both an HCV subgenomic replicon system and Huh7 cells expressing NS3. HCV NS3 protein inhibits WRN-mediated DNA repair and reduces the repair efficiency of nonhomologous end joining. It interferes with Ku70 recruitment to the double-strand break sites and alters the nuclear distribution of WRN-Ku repair complex. In addition, WRN is a substrate of the NS3/4A protease; the level of WRN protein is regulated by both the proteasome degradation pathway and HCV NS3/4A protease activity. The dual role of HCV NS3 and NS3/4A proteins in regulating the function and expression level of the WRN protein intensifies the effect of impairment on DNA repair. This may lead to an accumulation of DNA mutations and genome instability and, eventually, tumor development.IMPORTANCE HCV infection is a worldwide problem of public health and a major contributor to hepatocellular carcinoma. The single-stranded RNA virus with RNA-dependent RNA polymerase experiences a high error rate and develops strategies to escape the immune system and hepatocarcinogenesis. Studies have revealed the involvement of HCV proteins in the impairment of DNA repair. The present study aimed to further elucidate mechanisms by which the viral NS3 protein impairs the repair of DNA damage. Our results clearly indicate that HCV NS3/4A protease targets WRN for degradation, and, at the same time, diminishes the repair efficiency of nonhomologous end joining by interfering with the recruitment of Ku protein to the DNA double-strand break sites. The study describes a novel mechanism by which the NS3 protein influences DNA repair and provides new insight into the molecular mechanism of HCV pathogenesis.


Assuntos
Reparo do DNA por Junção de Extremidades , Hepacivirus/genética , Hepacivirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Helicase da Síndrome de Werner/metabolismo , Linhagem Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Células HEK293 , Hepatite C Crônica/genética , Humanos , Autoantígeno Ku/genética , RNA Polimerase Dependente de RNA/genética , Proteínas não Estruturais Virais/genética , Helicase da Síndrome de Werner/fisiologia
8.
Front Microbiol ; 10: 50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30761102

RESUMO

Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1ß) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1ß secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1ß secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K+ efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

9.
J Biomed Sci ; 25(1): 47, 2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29793506

RESUMO

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) consists of a positive-sense, single-stranded RNA genome and four structural proteins: the spike, envelope, membrane, and nucleocapsid protein. The assembly of the viral genome into virus particles involves viral structural proteins and is believed to be mediated through recognition of specific sequences and RNA structures of the viral genome. METHODS AND RESULTS: A culture system for the production of MERS coronavirus-like particles (MERS VLPs) was determined and established by electron microscopy and the detection of coexpressed viral structural proteins. Using the VLP system, a 258-nucleotide RNA fragment, which spans nucleotides 19,712 to 19,969 of the MERS-CoV genome (designated PS258(19712-19969)ME), was identified to function as a packaging signal. Assembly of the RNA packaging signal into MERS VLPs is dependent on the viral nucleocapsid protein. In addition, a 45-nucleotide stable stem-loop substructure of the PS258(19712-19969)ME interacted with both the N-terminal domain and the C-terminal domain of the viral nucleocapsid protein. Furthermore, a functional SARS-CoV RNA packaging signal failed to assemble into the MERS VLPs, which indicated virus-specific assembly of the RNA genome. CONCLUSIONS: A MERS-oV RNA packaging signal was identified by the detection of GFP expression following an incubation of MERS VLPs carrying the heterologous mRNA GFP-PS258(19712-19969)ME with virus permissive Huh7 cells. The MERS VLP system could help us in understanding virus infection and morphogenesis.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Montagem de Vírus/genética , Linhagem Celular Tumoral , Células HEK293 , Humanos , RNA Mensageiro/metabolismo
10.
J Formos Med Assoc ; 117(6): 471-479, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28549591

RESUMO

BACKGROUND/PURPOSE: LMBD1 protein, a type IV-B plasma membrane protein possessing nine putative trans-membrane domains, was previously demonstrated at cellular level to play a critical part in the signaling cascade of insulin receptor through its involvement in regulating clathrin-mediated endocytosis. However, at physiological level, the significance of LMBD1 protein in cardiac development remains unclear. METHODS: To understand the role of Lmbrd1 gene involved in the cardiac function, heterozygous knockout mice were used as an animal model system. The pathological outcomes were analyzed by micro-positron emission tomography, ECG acquisition, cardiac ultrasound, and immunohistochemistry. RESULTS: By studying the heterozygous knockout of Lmbrd1 (Lmbrd1+/-), we discovered that lack of Lmbrd1 not only resulted in the increase of cardiac-glucose uptake, pathological consequences were also observed. Here, we have distinguished that Lmbrd1+/- is sufficient in causing cardiac diseases through a pathway independent of the recessive vitamin B12 cblF cobalamin transport defect. Lmbrd1+/- mice exhibited an increase in myocardial glucose uptake and insulin receptor signaling that is insensitive to the administration of additional insulin. Pathological symptoms such as cardiac hypertrophy, ventricular tissue fibrosis, along with the increase of heart rate and cardiac muscle contractility were observed. As Lmbrd1+/- mice aged, the decrease in ejection fraction and fraction shortening showed signs of ventricular function deterioration. CONCLUSION: The results suggested that Lmbrd1 gene not only plays a significant role in mediating the energy homeostasis in cardiac tissue, it may also be a key factor in the regulation of cardiac function in mice.


Assuntos
Cardiomegalia/genética , Miócitos Cardíacos/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Receptor de Insulina/metabolismo , Alelos , Animais , Cardiomegalia/diagnóstico por imagem , Modelos Animais de Doenças , Ecocardiografia , Masculino , Camundongos , Camundongos Knockout , Tomografia por Emissão de Pósitrons , Transdução de Sinais
11.
J Biol Chem ; 291(50): 26226-26238, 2016 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-27807029

RESUMO

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro205 was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Núcleo Celular/metabolismo , Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Vírion/metabolismo , Montagem de Vírus/fisiologia , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Núcleo Celular/virologia , Células Hep G2 , Antígenos da Hepatite delta/genética , Humanos , Sinais de Localização Nuclear/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Peptídeos/farmacologia , Domínios Proteicos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Vírion/genética , Montagem de Vírus/efeitos dos fármacos
12.
J Biomed Sci ; 23: 14, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26801988

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex. RESULTS: A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein. CONCLUSIONS: A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.


Assuntos
Peptidil Dipeptidase A/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Vimentina/metabolismo , Internalização do Vírus , Enzima de Conversão de Angiotensina 2 , Animais , Chlorocebus aethiops , Peptidil Dipeptidase A/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Células Sf9 , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera , Células Vero , Vimentina/genética
13.
Liver Int ; 34(9): 1358-68, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25360475

RESUMO

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is highly associated with the type 2 diabetes mellitus, but the detailed mechanisms by which the viral proteins are involved in the clinical outcome remain unclear. METHODS: A cDNA microarray analysis was performed following introducing an NS5A-encoding plasmid or a control vector into a mouse system by hydrodynamics- based transfection. Differentially expressed genes that are associated with gluconeogenesis were selected and their expression levels in HCV patients, in NS5A-expressing systems, and in the viral subgenomic replicon system were further examined by real-time quantitative polymerase chain reaction and Western blot analysis. RESULTS: Differential gene expression including an upregulation of the gluconeogenic rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK) compared with controls was detected in mouse hepatocytes expressing HCV NS5A and in HCV patients with diabetes. In addition, an NS5A-dependent increase in glucose production was demonstrated in human primary hepatocytes. The upregulation of PEPCK and peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a) were also detected in NS5A-expressing cells and in the viral genotype 1b subgenomic replicon system. Further studies demonstrated that the NS5A-mediated upregulation of PEPCK and PGC-1a genes were resulted from the activation of PI3K-Akt and JNK signalling pathways. In addition, the expression levels of the forkhead transcription factor FoxO1 and the liver-enriched transcription factor HNF-4a were increased in HCV NS5A expressing cells. CONCLUSIONS: By upregulating the expression of PEPCK gene via its transactivators FoxO1 and HNF-4a, and the coactivator PGC-1a, the NS5A promotes the production of hepatic glucose which may contribute to the development of HCV-associated type 2 diabetes mellitus.


Assuntos
Diabetes Mellitus Tipo 2/virologia , Regulação da Expressão Gênica/fisiologia , Gluconeogênese/fisiologia , Hepacivirus/metabolismo , Hepatite C/complicações , Transdução de Sinais/fisiologia , Proteínas não Estruturais Virais/metabolismo , Animais , Western Blotting , Diabetes Mellitus Tipo 2/etiologia , Glucose/metabolismo , Hepatócitos/metabolismo , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Oncogênica v-akt/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
14.
J Biol Chem ; 288(45): 32424-32432, 2013 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-24078630

RESUMO

Energy homeostasis is crucial for maintaining normally functioning cells; disturbances in this balance often cause various diseases. The limb region 1 (LMBR1) domain containing 1 gene (lmbrd1) encodes the LMBD1 protein that possesses 9 putative transmembrane domains. LMBD1 has been suggested to be involved in the lysosome in aiding the export of cobalamin. In this study, we determined that LMBD1 plays a regulatory role in the plasma membrane. A micro-positron emission tomography analysis showed that a single-allele knock-out of lmbrd1 increased the (18)F-fluorodeoxyglucose uptake in murine hearts. In addition, the knockdown of lmbrd1 resulted in an up-regulated signaling of the insulin receptor (IR) and its downstream signaling molecule, Akt. Confocal and live total internal reflection fluorescence microscopy showed that LMBD1 co-localized and co-internalized with clathrin and the IR, but not with the transferrin receptor. The results of the mutation analysis and phenotypic rescue experiments indicate that LMBD1 interacts with adaptor protein-2 and is involved in the unique clathrin-mediated endocytosis of the IR. LMBD1 selectively interacts with the IR. The knockdown of lmbrd1 attenuated IR endocytosis, resulting in the perturbation of the IR recycling pathway and consequential enhancement of the IR signaling cascade. In summary, LMBD1 plays an imperative role in mediating and regulating the endocytosis of the IR.


Assuntos
Endocitose/fisiologia , Miocárdio/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/fisiologia , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Animais , Linhagem Celular , Clatrina/genética , Clatrina/metabolismo , Fluordesoxiglucose F18/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Mutantes , Proteínas de Transporte Nucleocitoplasmático/genética , Tomografia por Emissão de Pósitrons , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Compostos Radiofarmacêuticos/farmacologia , Ratos , Receptor de Insulina/genética
15.
J Virol ; 87(3): 1596-604, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23175358

RESUMO

Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.


Assuntos
Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , Interações Hospedeiro-Patógeno , Carioferinas/metabolismo , Lamina Tipo A/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Humanos , Imunoprecipitação , Multimerização Proteica , Montagem de Vírus , Proteína Exportina 1
16.
J Virol ; 84(15): 7703-12, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20484496

RESUMO

Severe acute respiratory syndrome coronavirus (SARS-CoV) was identified to be the causative agent of SARS with atypical pneumonia. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV. It is not clear whether ACE2 conveys signals from the cell surface to the nucleus and regulates expression of cellular genes upon SARS-CoV infection. To understand the pathogenesis of SARS-CoV, human type II pneumocyte (A549) cells were incubated with the viral spike protein or with SARS-CoV virus-like particles containing the viral spike protein to examine cytokine modulation in lung cells. Results from oligonucleotide-based microarray, real-time PCR, and enzyme-linked immunosorbent assays indicated an upregulation of the fibrosis-associated chemokine (C-C motif) ligand 2 (CCL2) by the viral spike protein and the virus-like particles. The upregulation of CCL2 by SARS-CoV spike protein was mainly mediated by extracellular signal-regulated kinase 1 and 2 (ERK1/2) and AP-1 but not the IkappaBalpha-NF-kappaB signaling pathway. In addition, Ras and Raf upstream of the ERK1/2 signaling pathway were involved in the upregulation of CCL2. Furthermore, ACE2 receptor was activated by casein kinase II-mediated phosphorylation in cells pretreated with the virus-like particles containing spike protein. These results indicate that SARS-CoV spike protein triggers ACE2 signaling and activates fibrosis-associated CCL2 expression through the Ras-ERK-AP-1 pathway.


Assuntos
Quimiocina CCL2/biossíntese , Glicoproteínas de Membrana/imunologia , Peptidil Dipeptidase A/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Transdução de Sinais , Proteínas do Envelope Viral/imunologia , Enzima de Conversão de Angiotensina 2 , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glicoproteína da Espícula de Coronavírus , Regulação para Cima
17.
FEBS Lett ; 584(3): 482-6, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19958771

RESUMO

Hepatitis C virus (HCV) internal non-structural protein 3 (NS3) cleavage can occur in trans in the presence of NS4A. In this study, we have further demonstrated a critical role of the helicase domain in the internal NS3 cleavage, different from HCV polyprotein processing which requires only the serine protease domain. The NTPase domain of NS3 helicase interacts with the RNA binding domain to facilitate internal NS3 cleavage. In addition, NS3 protease activity contributes to the transforming ability of the major internal cleavage product NS3(1-402). These findings imply important roles of the internal cleavage and protease activity of the NS3 protein in the pathogenesis of HCV.


Assuntos
Hepacivirus/enzimologia , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Hepacivirus/genética , Humanos , Camundongos , Células NIH 3T3 , Poliproteínas/genética , Poliproteínas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , RNA Helicases/química , RNA Helicases/genética , Serina Proteases/química , Serina Proteases/genética , Serina Proteases/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
18.
Cell Transplant ; 19(2): 231-43, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19906331

RESUMO

Oval cells and hepatocytes rarely proliferate simultaneously. This study aimed to determine the impacts of hepatocyte transplantation on the response and fate of oval cells that are activated to proliferate in acute severe hepatic injury. Retrorsine + D-galactosamine (R+D-gal) treatment was used to induce acute hepatic injury and to elicit extensive activation of oval cells in male dipeptidyl peptidase IV-deficient F344 rats. These rats were then randomized to receive wild-type hepatocyte transplantation or vehicle intraportally. The kinetics of oval cell response and their differentiation fate were analyzed. Results showed that oval cells were activated early and differentiated into hepatocytes in R+D-gal-treated rats without hepatocyte transplantation. With hepatocyte transplantation, the oval cells were recruited later and continued to proliferate in parallel with the massive proliferation of transplanted hepatocytes. They formed ductules and differentiated into biliary cells. When hepatocytes were transplanted at the day when oval cells were at their peak response, the numerous activated oval cells ceased to differentiate into hepatocytes and remained in ductular form. The ductular oval cells were capable of differentiating into hepatocytes again when the donor hepatocytes were inhibited to proliferate. We conclude that hepatocyte transplantation changes the mechanism of liver reconstitution and affects the differentiation fate of host oval cells in acute severe hepatic injury.


Assuntos
Diferenciação Celular , Doença Hepática Induzida por Substâncias e Drogas , Hepatócitos/transplante , Fígado/lesões , Animais , Antineoplásicos Fitogênicos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/cirurgia , Dipeptidil Peptidase 4/deficiência , Galactosamina/toxicidade , Hepatócitos/citologia , Humanos , Fígado/citologia , Masculino , Alcaloides de Pirrolizidina/toxicidade , Distribuição Aleatória , Ratos , Ratos Endogâmicos F344
19.
J Virol ; 83(23): 12314-24, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19793827

RESUMO

Clathrin is involved in the endocytosis and exocytosis of cellular proteins and the process of virus infection. We have previously demonstrated that large hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis delta virus (HDV) are not clear. In this study, we found that clathrin heavy chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a single amino acid substitution at the clathrin box retained nuclear export activity but failed to interact with CHC and to assemble into virus-like particles. Downregulation of CHC function by a dominant-negative mutant or by short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion of hepatitis B virus subviral particles. In addition, the coexistence of a cell-permeable peptide derived from the C terminus of HDAg-L significantly interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and CHC were found to colocalize with the trans-Golgi network and were highly enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which assembles less efficiently than genotype I HDV does, has a putative clathrin box in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of the various HDV genotypes correlates well with the CHC-binding activity of their HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin box and the nuclear export signal at the C terminus of HDAg-L are potential new molecular targets for HDV therapy.


Assuntos
Cadeias Pesadas de Clatrina/metabolismo , Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , Montagem de Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Antígenos da Hepatite delta/genética , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Mutação Puntual , Ligação Proteica , Transporte Proteico
20.
Hepatology ; 50(4): 1184-93, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19676133

RESUMO

UNLABELLED: Liver cirrhosis is characterized by progressive accumulation of extracellular matrix following chronic liver injuries. In the extracellular space, the constant turnover of liver matrix is regulated by the matrix metalloproteinase (MMP) class of enzyme. To assess whether genetic variations in MMP would result in diversity of liver cirrhosis, a case-control study of 320 patients with hepatocellular carcinoma, with or without cirrhosis, was conducted. Ten single-nucleotide polymorphism markers from four potential fibrosis-associated genes were selected for genotyping. Among these genes, a nonsynonymous single-nucleotide polymorphism which generates the variation of Gly-137 and Asp-137 in the MMP-7 gene was found to be strongly associated with the development of liver cirrhosis. In contrast to MMP-7(Gly-137) that predominantly secretes out into the cell culture medium, the cirrhosis-associated MMP-7(Asp-137) variant is preferentially localized on the extracellular membranes where it exerts its proteolytic activity on pericellular substrates. Functional analysis demonstrated an increased ability of the MMP-7(Asp-137) variant to associate with the cell surface CD151 molecule. In wound-healing and Boyden chamber assays, cell motility was specifically enhanced with the expression of MMP-7(Asp-137) as compared to the cells expressing MMP-7(Gly-137). These results demonstrate that the MMP-7(Asp-137) variant confers a gain-of-function phenotype for MMP-7. CONCLUSION: We have identified a novel genetic association of MMP-7(Asp-137) variant with liver cirrhosis in patients with hepatocellular carcinoma. Whether the MMP-7 variant can be a new marker for liver cirrhosis will be further studied.


Assuntos
Carcinoma Hepatocelular/metabolismo , Predisposição Genética para Doença/genética , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Adulto , Idoso , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Membrana Celular/metabolismo , Feminino , Genótipo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Tetraspanina 24
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