RESUMEN
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.
Asunto(s)
Citoesqueleto/fisiología , Microtúbulos/fisiología , Mitosis , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/fisiología , Tubulina (Proteína)/química , Animales , Ciclo Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transporte Nucleocitoplasmático/genética , Dominios y Motivos de Interacción de Proteínas , Huso Acromático/fisiologíaRESUMEN
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.
Asunto(s)
Infecciones por Coronavirus/metabolismo , Infecciones por Coronavirus/virología , Proteína 58 DEAD Box/metabolismo , Interferón Tipo I/biosíntesis , Interferones/biosíntesis , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Proteínas de la Nucleocápside/metabolismo , Transducción de Señal , Proteínas Adaptadoras de Señalización CARD/metabolismo , Línea Celular , Infecciones por Coronavirus/genética , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferones/genética , Regiones Promotoras Genéticas , Unión Proteica , Receptores Inmunológicos , Factores de Transcripción , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas , Interferón lambdaRESUMEN
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.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Hepacivirus/genética , Hepacivirus/metabolismo , Proteínas no Estructurales Virales/metabolismo , Helicasa del Síndrome de Werner/metabolismo , Línea Celular , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Células HEK293 , Hepatitis C Crónica/genética , Humanos , Autoantígeno Ku/genética , ARN Polimerasa Dependiente del ARN/genética , Proteínas no Estructurales Virales/genética , Helicasa del Síndrome de Werner/fisiologíaRESUMEN
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.
Asunto(s)
Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Proteínas de la Nucleocápside/metabolismo , ARN Viral/metabolismo , Ensamble de Virus/genética , Línea Celular Tumoral , Células HEK293 , Humanos , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Cardiomegalia/genética , Miocitos Cardíacos/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Receptor de Insulina/metabolismo , Alelos , Animales , Cardiomegalia/diagnóstico por imagen , Modelos Animales de Enfermedad , Ecocardiografía , Masculino , Ratones , Ratones Noqueados , Tomografía de Emisión de Positrones , Transducción de SeñalRESUMEN
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.
Asunto(s)
Peptidil-Dipeptidasa A/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Vimentina/metabolismo , Internalización del Virus , Enzima Convertidora de Angiotensina 2 , Animales , Chlorocebus aethiops , Peptidil-Dipeptidasa A/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Células Sf9 , Glicoproteína de la Espiga del Coronavirus/genética , Spodoptera , Células Vero , Vimentina/genéticaRESUMEN
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.
Asunto(s)
Endocitosis/fisiología , Miocardio/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Complejo 2 de Proteína Adaptadora/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Línea Celular , Clatrina/genética , Clatrina/metabolismo , Fluorodesoxiglucosa F18/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Mutantes , Proteínas de Transporte Nucleocitoplasmático/genética , Tomografía de Emisión de Positrones , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Radiofármacos/farmacología , Ratas , Receptor de Insulina/genéticaRESUMEN
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.
Asunto(s)
Virus de la Hepatitis Delta/fisiología , Antígenos de Hepatitis delta/metabolismo , Interacciones Huésped-Patógeno , Carioferinas/metabolismo , Lamina Tipo A/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Humanos , Inmunoprecipitación , Multimerización de Proteína , Ensamble de Virus , Proteína Exportina 1RESUMEN
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.
Asunto(s)
Diabetes Mellitus Tipo 2/virología , Regulación de la Expresión Génica/fisiología , Gluconeogénesis/fisiología , Hepacivirus/metabolismo , Hepatitis C/complicaciones , Transducción de Señal/fisiología , Proteínas no Estructurales Virales/metabolismo , Animales , Western Blotting , Diabetes Mellitus Tipo 2/etiología , Glucosa/metabolismo , Hepatocitos/metabolismo , Humanos , MAP Quinasa Quinasa 4/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína Oncogénica v-akt/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , TransfecciónRESUMEN
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.
Asunto(s)
Infecciones por Coronavirus , Coronavirus del Síndrome Respiratorio de Oriente Medio , Vacunas Virales , Animales , Ratones , Humanos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Dipeptidil Peptidasa 4 , Inmunidad Celular , Ratones Transgénicos , Adyuvantes Inmunológicos , Proteínas Recombinantes , Vacunas de Subunidad , Glicoproteína de la Espiga del CoronavirusRESUMEN
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.
Asunto(s)
Quimiocina CCL2/biosíntesis , Glicoproteínas de Membrana/inmunología , Peptidil-Dipeptidasa A/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Transducción de Señal , Proteínas del Envoltorio Viral/inmunología , Enzima Convertidora de Angiotensina 2 , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/virología , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glicoproteína de la Espiga del Coronavirus , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Cadenas Pesadas de Clatrina/metabolismo , Virus de la Hepatitis Delta/fisiología , Antígenos de Hepatitis delta/metabolismo , Ensamble de Virus , Animales , Línea Celular , Chlorocebus aethiops , Cadenas Pesadas de Clatrina/antagonistas & inhibidores , Antígenos de Hepatitis delta/genética , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Mutación Puntual , Unión Proteica , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Predisposición Genética a la Enfermedad/genética , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Adulto , Anciano , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Membrana Celular/metabolismo , Femenino , Genotipo , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Factores de Riesgo , Tetraspanina 24RESUMEN
The open reading frame 3 of the severe acute respiratory syndrome coronavirus (SARS-CoV) genome encodes a predicted protein 3a, consisting of 274 amino acids, that lacks any significant similarities to any known protein. We generated specific antibodies against SARS protein 3a by using a synthetic peptide (P2) corresponding to amino acids 261-274 of the putative protein. Anti-P2 antibodies and the sera from SARS patients could specifically detect the recombinant SARS protein 3a expressed in Escherichia coli and in Vero E6 cells. Expression of SARS protein 3a was detected at 8-12 h after infection and reached a higher level after approximately 24 h in SARS-CoV-infected Vero E6 cells. Protein 3a was also detected in the alveolar lining pneumocytes and some intra-alveolar cells of a SARS-CoV-infected patient's lung specimen. Recombinant protein 3a expressed in Vero E6 cells and protein 3a in the SARS-CoV-infected cells was distributed over the cytoplasm in a fine punctate pattern with partly concentrated staining in the Golgi apparatus. Our study demonstrates that SARS-CoV indeed expresses a novel protein 3a, which is present only in SARS-CoV and not in other known CoVs.
Asunto(s)
Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Proteínas Virales/química , Animales , Anticuerpos/química , Chlorocebus aethiops , Clonación Molecular , Grupo Citocromo b/química , Citoplasma/metabolismo , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Aparato de Golgi/metabolismo , Humanos , Inmunohistoquímica , Pulmón/virología , Microscopía Fluorescente , Nucleocápside/metabolismo , Sistemas de Lectura Abierta , Péptidos/química , Estructura Terciaria de Proteína , Alveolos Pulmonares/virología , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Células Vero , Proteínas del Envoltorio Viral , Proteínas ViroporinasRESUMEN
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.
Asunto(s)
Hepacivirus/enzimología , ARN Helicasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Hepacivirus/genética , Humanos , Ratones , Células 3T3 NIH , Poliproteínas/genética , Poliproteínas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , ARN Helicasas/química , ARN Helicasas/genética , Serina Proteasas/química , Serina Proteasas/genética , Serina Proteasas/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaRESUMEN
Hepatitis C virus often causes persistent infection and hepatocellular carcinoma. Studies have demonstrated the roles of viral nonstructural protein 5A (NS5A) in the induction of chromosome aneuploidy, but the molecular mechanisms are not clear. In this study, hydrodynamics-based in vivo transfection was applied to a mouse system. Mouse hepatocytes that successfully expressed NS5A protein were isolated by laser capture microdissection. Gene expression profiles of the NS5A-expressing hepatocytes were examined by an Affymetrix oligonucleotide microarray system. Aspm (abnormal spindle-like, microcephaly associated), which encodes the mitotic spindle protein ASPM, was identified to be differentially expressed in the absence and the presence of NS5A. The down-regulation of Aspm mRNA and ASPM protein was confirmed by real time polymerase chain reaction and Western blot analysis, respectively, both in mouse model systems and in viral subgenomic replicon and in vitro transfection culturing systems. In addition, cultured cells that constitutively expressed NS5A protein showed G(2)/M cell cycle block and chromosome aneuploidy. Overexpression of ASPM relieved the G(2)/M cell cycle block. Furthermore, NS5A protein repressed the promoter activity of Aspm gene in a dose-dependent manner. The regulatory effect was abolished when amino acid substitutions P2209L, T2214A, and T2217G known to interrupt the NS5A-PKR interaction were introduced into the NS5A protein. This indicates that the down-regulation of Aspm expression is via the PKR-p38 signaling pathway. These results suggest that NS5A protein down-regulates the expression of the mitotic spindle protein ASPM and induces aberrant mitotic cell cycle associated with chromosome instability and hepatocellular carcinoma.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Regulación Viral de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/fisiología , eIF-2 Quinasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Células COS , Proteínas de Unión a Calmodulina , Chlorocebus aethiops , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIHRESUMEN
Clathrin-mediated endocytosis is a common pathway for viral entry, but little is known about the direct association of viral protein with clathrin in the cytoplasm. In this study, a putative clathrin box known to be conserved in clathrin adaptors was identified at the C terminus of the large hepatitis delta antigen (HDAg-L). Similar to clathrin adaptors, HDAg-L directly interacted with the N terminus of the clathrin heavy chain through the clathrin box. HDAg-L is a nucleocytoplasmic shuttle protein important for the assembly of hepatitis delta virus (HDV). Here, we demonstrated that brefeldin A and wortmannin, inhibitors of clathrin-mediated exocytosis and endosomal trafficking, respectively, specifically blocked HDV assembly but had no effect on the assembly of the small surface antigen of hepatitis B virus. In addition, cytoplasm-localized HDAg-L inhibited the clathrin-mediated endocytosis of transferrin and the degradation of epidermal growth factor receptor. These results indicate that HDAg-L is a new clathrin adaptor-like protein, and it may be involved in the maturation and pathogenesis of HDV coinfection or superinfection with hepatitis B virus through interaction with clathrin.
Asunto(s)
Cadenas Pesadas de Clatrina/química , Virus de la Hepatitis Delta/química , Antígenos de Hepatitis delta/química , Animales , Células COS , Chlorocebus aethiops , Cadenas Pesadas de Clatrina/metabolismo , Virus de la Hepatitis Delta/metabolismo , Antígenos de Hepatitis delta/metabolismo , HumanosRESUMEN
The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT(402)|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.
Asunto(s)
Proteínas Portadoras/metabolismo , Hepacivirus/metabolismo , Poliproteínas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Proteínas Virales/genéticaRESUMEN
The genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS3 and NS2-3 proteases. Nonstructural protein 4A (NS4A) is a cofactor of the NS3 serine protease and has been demonstrated to inhibit protein synthesis. In this study, GST pull-down assay was performed to examine potential cellular factors that interact with the NS4A protein and are involved in the pathogenesis of HCV. A trypsin digestion followed by LC-MS/MS analysis revealed that one of the GST-NS4A-interacting proteins to be eukaryotic elongation factor 1A (eEF1A). Both the N-terminal domain of NS4A from amino acid residues 1-20, and the central domain from residues 21-34 interacted with eEF1A, but the central domain was the key player involved in the NS4A-mediated translation inhibition. NS4A(21-34) diminished both cap-dependent and HCV IRES-mediated translation in a dose-dependent manner. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. Taken together, NS4A inhibits host and viral translation through interacting with eEF1A, implying a possible mechanism by which NS4A is involved in the pathogenesis and chronic infection of HCV.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor 1 de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , Caperuzas de ARN , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Cartilla de ADN , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas no Estructurales Virales , Proteínas Virales/químicaRESUMEN
The process of host factor-mediated nucleocytoplasmic transport is critical for diverse cellular events in eukaryotes and the life cycle of viruses. We have previously identified a chromosome region maintenance 1-independent nuclear export signal (NES) at the C terminus of the large form of hepatitis delta antigen (HDAg), designated NES(HDAg-L) that is required for the assembly of hepatitis delta virus (HDV) (C.-H. Lee et al., J. Biol. Chem. 276:8142-8148, 2001). To look for interacting proteins of the NES(HDAg-L), yeast two-hybrid screening was applied using the GAL4-binding domain fused to the NES(HDAg-L) as bait. Among the positive clones, one encodes a protein, designated NESI [NES(HDAg-L) interacting protein] that specifically interacted with the wild-type NES(HDAg-L) but not with the export/package-defective HDAg-L mutant, NES*(HDAg-L), in which Pro-205 has been replaced by Ala. Northern blot analysis revealed NESI as the gene product of a 1.9-kb endogenous mRNA transcript that is present predominantly in human liver tissue. NESI consists of 467 amino acid residues and bears a putative actin-binding site and a bipartite nuclear localization signal. Specific interaction between HDAg-L and NESI was further confirmed by coimmunoprecipitation and immunofluorescence staining. Overexpression of antisense NESI RNAs inhibited the expression of NESI and abolished HDAg-L-mediated nuclear export and assembly of HDV genomic RNA. These data indicate a critical role of NESI in the assembly of HDV through interaction with HDAg-L.