RESUMEN
Orf virus (ORFV) infects sheep and goat tissues, resulting in severe proliferative lesions. To analyze cellular protein expression in ORFV-infected goat skin fibroblast (GSF) cells, we used two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ). The proteomics approach was used along with quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect differentially expressed proteins in ORFV-infected GSF cells and mock-infected GSF cells. A total of 282 differentially expressed proteins were identified. It was found that 222 host proteins were upregulated and 60 were downregulated following viral infection. We confirmed that these proteins were differentially expressed and found that heat shock 70-kDa protein 1B (HSPA1B) was differentially expressed and localized in the cytoplasm. It was also noted that HSPA1B caused inhibition of viral proliferation, in the middle and late stages of viral infection. The differentially expressed proteins were associated with the biological processes of viral binding, cell structure, signal transduction, cell adhesion, and cell proliferation.
Asunto(s)
Fibroblastos/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Virus del Orf/fisiología , Proteoma/genética , Replicación Viral , Animales , Células Cultivadas , Cromatografía Liquida , Fibroblastos/virología , Cabras , Interacciones Huésped-Patógeno , Virus del Orf/genética , Proteómica , Espectrometría de Masas en TándemRESUMEN
As a zoonotic disease, ovine contagious pustular dermatitis (Orf) is a serious threat to sheep as well as humans. Orf virus (ORFV) interferon resistance protein (VIR) is the principal virulence protein that encodes a dsRNA-binding protein to inhibit host antiviral response. p53 is one of the key proteins of the host antiviral innate immunity. It not only enhances type I interferon secretion but also induces apoptosis in infected cells, and plays a crucial role in the immune response against various viral infections. However, it remains to be elucidated what role p53 plays in ORFV replication and whether ORFV's own protein VIR regulates p53 expression to promote self-replication. In this study, we showed that p53 has an antiviral effect on ORFV and can inhibit ORFV replication. In addition, ORFV nonstructural protein VIR interacts with p53 and degrades p53, which inhibits p53-mediated positive regulation of downstream antiviral genes. This study provides new insight into the immune evasion mediated by ORFV and identifies VIR as an antagonistic factor for ORFV to evade the antiviral response.
Asunto(s)
Interacciones Microbiota-Huesped/genética , Virus del Orf/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Virales/genética , Replicación Viral/genética , Animales , Línea Celular , Cricetinae , Ectima Contagioso/virología , Fibroblastos/inmunología , Fibroblastos/virología , Regulación Viral de la Expresión Génica , Cabras , Evasión Inmune/genética , Inmunidad Innata , Riñón/citología , Virus del Orf/fisiología , Ovinos , Piel/citología , Proteínas Virales/metabolismoRESUMEN
The components of foot-and-mouth disease virus (FMDV) interact with host cellular proteins to promote self-replication and evade the host immune response. Previous studies have shown that FMDV 3A, 2C and 2B proteins interact with host cellular proteins involved in FMDV replication. However, whether the other host proteins have an impact on FMDV replication is less understood. In this study, we identified DDX56 as a positive regulator of FMDV replication. DDX56 overexpression increased FMDV replication, whereas DDX56 knockdown had the opposite effect. DDX56 interacted and cooperated with FMDV 3A to inhibit the type I interferon by reducing the phosphorylation of IRF3. Moreover, the D166 site of DDX56 played a role in increasing FMDV replication and cooperating with FMDV 3A to inhibit the phosphorylation of IRF3. Additionally, knockdown of DDX56 or FMDV 3A results also showed that DDX56 cooperated with FMDV 3A to inhibit the phosphorylation of IRF3. These results suggest that the interaction between FMDV 3A protein and the host protein DDX56 is critical for FMDV replication.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Virus de la Fiebre Aftosa/fisiología , Fiebre Aftosa/virología , Interacciones Microbiota-Huesped , Factor 3 Regulador del Interferón/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , Animales , Células HEK293 , Hepatocitos , Humanos , Interferón Tipo I/metabolismo , Fosforilación , Unión Proteica , PorcinosRESUMEN
Tumor progression locus 2 (TPL2) is a serine/threonine kinase that belongs to the MAP3K family. The activated TPL2 regulates the innate immune-relevant signaling pathways, such as ERK, JNK, and NF-κB, and the differentiation of immune cells, for example, CD4+ T and NK cells. Therefore, TPL2 plays a critical role in regulating the innate immune response. The present review summarizes the recent advancements in the TPL2-regulated innate immune response.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Diferenciación Celular , Quimiocinas/metabolismo , Humanos , Inmunidad Innata , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas , Activación de Macrófagos , Activación Neutrófila , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Nucleoside diphosphate kinase 1 (NME1) is well-known as a tumor suppressor that regulates p53 function to prevent cancer metastasis and progression. However, the role of NME1 in virus-infected cells remains unknown. Here, we showed that NME1 suppresses viral replication in foot-and-mouth disease virus (FMDV)-infected cells. NME1-enhanced p53-mediated transcriptional activity and induction of interferon-inducible antiviral genes expression. FMDV infection decreased NME1 protein expression. The 2B and VP4 proteins were identified as the viral factors that induced reduction of NME1. FMDV 2B protein has a suppressive effect on host protein expression. We measured, for the first time, VP4-induced lysosomal degradation of host protein; VP4-induced degradation of NME1 through the macroautophagy pathway, and impaired p53-mediated signaling. p53 plays significant roles in antiviral innate immunity by inducing several interferon-inducible antiviral genes expression, such as, ISG20, IRF9, RIG-I, and ISG15. VP4 promoted interaction of p53 with murine double minute 2 (MDM2) through downregulation of NME1 resulting in destabilization of p53. Therefore, 5-flurouracil-induced upregulation of ISG20, IRF9, RIG-I, and ISG15 were suppressed by VP4. VP4-induced reduction of NME1 was not related to the well-characterized blocking effect of FMDV on cellular translation, and no direct interaction was detected between NME1 and VP4. The 15-30 and 75-85 regions of VP4 were determined to be crucial for VP4-induced reduction of NME1. Deletion of these VP4 regions also inhibited the suppressive effect of VP4 on NME1-enhanced p53 signaling. In conclusion, these data suggest an antiviral role of NME1 by regulation of p53-mediated antiviral innate immunity in virus-infected cells, and reveal an antagonistic mechanism of FMDV that is mediated by VP4 to block host innate immune antiviral response.
Asunto(s)
Antivirales/inmunología , Virus de la Fiebre Aftosa/inmunología , Fiebre Aftosa/inmunología , Regulación de la Expresión Génica/inmunología , Interferones/inmunología , Lisosomas/inmunología , Nucleósido Difosfato Quinasas NM23/inmunología , Proteína p53 Supresora de Tumor/inmunología , Animales , Línea Celular , Regulación hacia Abajo/inmunología , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/inmunología , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunología , Proteínas Virales/inmunología , Replicación Viral/inmunologíaRESUMEN
Flavin-containing monooxygenase 3 (FMO3) plays a critical role in catalyzing the conversion of trimethylamine (TMA) to trimethylamine-N-oxide (TMAO) in vivo. Despite the well-documented association between FMO3 mutations and a 'fishy' off-flavor eggs in chicken and quail, little information is available regarding the molecular characteristic of goose (Anser cygnoides) FMO3 and its relationship with the yolk TMA content. To fill these gaps, we cloned the full-length cDNA sequence of goose FMO3, which comprised 1851bp encoding 531 amino acids. FMO3 mRNA was dramatically expressed in liver than in other tissues in the geese. Eight single nucleotide polymorphisms (SNPs) were detected in the entire coding region. The CC genotype at the T669C site, GG at the A723G site, and AA at the G734A site of FMO3 were highly significantly associated with elevated TMA content in goose egg yolk (P<0.001). Carriers of the A allele of G734A or C allele of T885C had yolk TMA content that had a high probability of being elevated after feeding with additional choline chloride (P=0.0429, OR=4.1300, 95%CI=1.0390-16.4270, and P=0.0251, OR=4.6060, 95%CI=1.1620-18.2620, respectively). This work lays a foundation for studying the function of FMO3 and yolk TMA content in goose. However, studies using larger sample sizes and more goose breeds are required to determine whether the fishy off-flavor trait exists in goose.
Asunto(s)
Proteínas Aviares/genética , Yema de Huevo/metabolismo , Gansos/genética , Metilaminas/metabolismo , Oxigenasas/genética , Polimorfismo Genético , Animales , Proteínas Aviares/metabolismo , Clonación Molecular , Huevos/análisis , Huevos/normas , Mutación Missense , Oxigenasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The alterations of MicroRNAs(miRNAs) in host cell after foot-and-mouth disease virus (FMDV) infection is still obscure. To increase our understanding of the pathogenesis of FMDV at the post-transcriptional regulation level, Solexa high-throu MicroRNAs (miRNAs) play an important role both in the post-transcriptional regulation of gene expression and host-virus interactions. Despite investigations of miRNA expression ghput sequencing and bioinformatic tools were used to identify differentially expressed miRNAs and analyze their functions during FMDV infection of PK-15 cells. Results indicated that 9,165,674 and 9,230,378 clean reads were obtained, with 172 known and 72 novel miRNAs differently expressed in infected and uninfected groups respectively. Some of differently expressed miRNAs were validated using stem-loop real-time quantitative RT-PCR. The GO annotation and KEGG pathway analysis for target genes revealed that differently expressed miRNAs were involved in immune response and cell death pathways.
Asunto(s)
Virus de la Fiebre Aftosa/fisiología , MicroARNs/genética , Animales , Línea Celular , Biología Computacional/métodos , Fiebre Aftosa/genética , Fiebre Aftosa/virología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Biblioteca de Genes , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Reproducibilidad de los ResultadosRESUMEN
The purpose of this study was to obtain immunogenic proteins and potential proteins of interest that were isolated from Mycoplasma capricolum subsp. capripneumoniae (Mccp) by MALDI-TOF mass spectrometry. One-dimensional SDS-PAGE and two-dimensional gel electrophoresis of whole cell preparation were conducted, and membrane proteome maps were prepared by immunoblotting. One-dimensional SDS-PAGE identified three immunogenic proteins with molecular masses in the range 29-97.2 kDa, two of which were in the membrane protein fraction. After two-dimensional gel electrophoresis, 20 highly immunogenic proteins were identified in the whole cell protein preparation while 9 immunogenic proteins were identified in the membrane protein fraction. This indicated that membrane proteins were the principle immunogenic proteins in Mccp. These proteins may have potential for the development of improved diagnostic tests and possible vaccines.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Mycoplasma capricolum/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Biología Computacional , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Proteínas de la Membrana/inmunología , Mycoplasma capricolum/inmunología , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
BACKGROUND: Rabies virus (RABV) can infect many different species of warm-blooded animals. Glycoprotein G plays a key role in viral pathogenicity and neurotropism, and includes antigenic domains that are responsible for membrane fusion and host cell receptor recognition. CASE PRESENTATION: A case of buffalo rabies in China was diagnosed by direct fluorescent antibody test, G gene reverse-transcriptase polymerase chain reaction, and RABV mouse inoculation test. Molecular characterization of the RABV was performed using DNA sequencing, phylogenetic analysis and amino acid sequence comparison based on the G gene from different species of animals. CONCLUSION: The results confirmed that the buffalo with suspected rabies was infected by RABV, which was genetically closely related to HNC (FJ602451) that was isolated from cattle in China in 2007. Comparison of the G gene among different species of animal showed that there were almost no amino acid changes among RABVs isolated from the same species of animals that distributed in a near region. However, there were many changes among RABVs that were isolated from different species of animal, or the same species from different geographic regions. This is believed to be the first case report of buffalo rabies in China, and the results may provide further information to understand the mechanism by which RABV breaks through the species barrier.