RESUMEN
Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.
Asunto(s)
Antivirales , Virus de la Fiebre Porcina Clásica , Dihidroorotato Deshidrogenasa , Proteínas no Estructurales Virales , Replicación Viral , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Línea Celular , Peste Porcina Clásica/tratamiento farmacológico , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/efectos de los fármacos , Virus de la Fiebre Porcina Clásica/crecimiento & desarrollo , Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/metabolismo , Dihidroorotato Deshidrogenasa/metabolismo , Relación Dosis-Respuesta a Droga , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Inmunoprecipitación , Microscopía Confocal , Mitocondrias/enzimología , Mitocondrias/metabolismo , ARN Viral/biosíntesis , Transducción de Señal/efectos de los fármacos , Porcinos/virología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Endopeptidasas , Histona Desacetilasa 1 , Inmunidad Innata , Complejo de la Endopetidasa Proteasomal , Factor de Transcripción Sp1 , Proteínas Virales , Animales , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/enzimología , Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/metabolismo , Virus de la Fiebre Porcina Clásica/patogenicidad , Endopeptidasas/química , Endopeptidasas/metabolismo , Histona Desacetilasa 1/biosíntesis , Histona Desacetilasa 1/metabolismo , Factor 3 Regulador del Interferón , Proteínas de la Nucleocápside/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factor de Transcripción Sp1/metabolismo , Porcinos/virología , Proteínas del Núcleo Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Ubiquitinas/metabolismo , Citocinas/metabolismo , Virus de la Diarrea Epidémica Porcina/inmunología , Virus de la Diarrea Epidémica Porcina/metabolismo , Dominios ProteicosRESUMEN
Classical swine fever virus (CSFV), which is a positive-sense, single-stranded RNA virus with an envelope, is a member of the Pestivirus genus in the Flaviviridae family. CSFV causes a severe and highly contagious disease in pigs and is prevalent worldwide, threatening the pig farming industry. The detailed mechanisms of the CSFV life cycle have been reported, but are still limited. Some receptors and attachment factors of CSFV, including heparan sulfate (HS), laminin receptor (LamR), complement regulatory protein (CD46), MER tyrosine kinase (MERTK), disintegrin, and metalloproteinase domain-containing protein 17 (ADAM17), were identified. After attachment, CSFV internalizes via clathrin-mediated endocytosis (CME) and/or caveolae/raft-dependent endocytosis (CavME). After internalization, CSFV moves to early and late endosomes before uncoating. During this period, intracellular trafficking of CSFV relies on components of the endosomal sorting complex required for transport (ESCRT) and Rab proteins in the endosome dynamics, with a dependence on the cytoskeleton network. This review summarizes the data on the mechanisms of CSFV attachment, internalization pathways, and intracellular trafficking, and provides a general view of the early events in the CSFV life cycle.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Pestivirus , Animales , Porcinos , Virus de la Fiebre Porcina Clásica/metabolismo , EndocitosisRESUMEN
The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation, assembly of infectious virus particles, and regulation of cellular signaling pathways. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism involved has not been elucidated. Here, we reported that NS5A directly interacted with NF-κB essential modulator (NEMO), a regulatory subunit of the IκB kinase (IKK) complex, to inhibit the NF-κB signaling pathway. Further investigations showed that the zinc finger domain of NEMO and the aa 126-250 segment of NS5A are essential for the interaction between NEMO and NS5A. Mechanistic analysis revealed that NS5A mediated the proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced the K27-linked but not the K48-linked polyubiquitination of NEMO for proteasomal degradation. In addition, NS5A blocked the K63-linked polyubiquitination of NEMO, thus inhibiting IKK phosphorylation, IκBα degradation, and NF-κB activation. These findings revealed a novel mechanism by which CSFV inhibits host innate immunity, which might guide the drug design against CSFV in the future.
Asunto(s)
Virus de la Fiebre Porcina Clásica , FN-kappa B , Animales , Porcinos , FN-kappa B/metabolismo , Virus de la Fiebre Porcina Clásica/metabolismo , Transducción de Señal , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Inmunidad InnataRESUMEN
Foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) possess positive-sense single-stranded RNA genomes and an internal ribosomal entry site (IRES) element within their 5'-untranslated regions. To investigate the common host factors associated with these IRESs, we established cell lines expressing a bicistronic luciferase reporter plasmid containing an FMDV-IRES or CSFV-IRES element between the Renilla and firefly luciferase genes. First, we treated FMDV-IRES cells with the French maritime pine extract, Pycnogenol (PYC), and examined its suppressive effect on FMDV-IRES activity, as PYC has been reported to have antiviral properties. Next, we performed microarray analysis to identify the host factors that modified their expression upon treatment with PYC, and confirmed their function using specific siRNAs. We found that polycystic kidney disease 1-like 3 (PKD1L3) and ubiquitin-specific peptidase 31 (USP31) were associated with FMDV-IRES activity. Moreover, silencing of these factors significantly suppressed CSFV-IRES activity. Thus, PKD1L3 and USP31 are host factors associated with the functions of FMDV- and CSFV-IRES elements.
Asunto(s)
Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica , Virus de la Fiebre Aftosa , Fiebre Aftosa , Animales , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/metabolismo , Virus de la Fiebre Aftosa/genética , Sitios Internos de Entrada al Ribosoma , PorcinosRESUMEN
As the important molecular machinery for membrane protein sorting in eukaryotic cells, the endosomal sorting and transport complexes (ESCRT-0/I/II/III and VPS4) usually participate in various replication stages of enveloped viruses, such as endocytosis and budding. The main subunit of ESCRT-I, Tsg101, has been previously revealed to play a role in the entry and replication of classical swine fever virus (CSFV). However, the effect of the whole ESCRT machinery during CSFV infection has not yet been well defined. Here, we systematically determine the effects of subunits of ESCRT on entry, replication, and budding of CSFV by genetic analysis. We show that EAP20 (VPS25) (ESCRT-II), CHMP4B and CHMP7 (ESCRT-III) regulate CSFV entry and assist vesicles in transporting CSFV from Clathrin, early endosomes, late endosomes to lysosomes. Importantly, we first demonstrate that HRS (ESCRT-0), VPS28 (ESCRT-I), VPS25 (ESCRT-II) and adaptor protein ALIX play important roles in the formation of virus replication complexes (VRC) together with CHMP2B/4B/7 (ESCRT-III), and VPS4A. Further analyses reveal these subunits interact with CSFV nonstructural proteins (NS) and locate in the endoplasmic reticulum, but not Golgi, suggesting the role of ESCRT in regulating VRC assembly. In addition, we demonstrate that VPS4A is close to lipid droplets (LDs), indicating the importance of lipid metabolism in the formation of VRC and nucleic acid production. Altogether, we draw a new picture of cellular ESCRT machinery in CSFV entry and VRC formation, which could provide alternative strategies for preventing and controlling the diseases caused by CSFV or other Pestivirus.
Asunto(s)
Virus de la Fiebre Porcina Clásica/metabolismo , Peste Porcina Clásica/virología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Virus de la Fiebre Porcina Clásica/genética , Clatrina/metabolismo , Retículo Endoplásmico/metabolismo , Interacciones Microbiota-Huesped , Porcinos , Vesículas Transportadoras , Internalización del Virus , Replicación ViralRESUMEN
Classical swine fever (CSF) is an economically important disease of pigs caused by classical swine fever virus (CSFV). The live attenuated vaccine C-strain (also called HCLV strain) against CSF was produced by multiple passages of a highly virulent strain in rabbits. However, the molecular determinants for its attenuation and protection remain unclear. In this study, we identified a unique glycosylation at position 986 (986NYT988) on the E2 glycoprotein Domain IV of C-strain but not (986NYA988) the highly virulent CSFV Shimen strain. We evaluated the infectivity, virulence, and protective efficacy of the C-strain-based mutant rHCLV-T988A lacking the glycosylation and Shimen strain mutant rShimen-A988T acquiring an additional glycosylation at position 986. rShimen-A988T showed a significantly decreased viral replication ability in SK6 cells, while rHCLV-T988A exhibited a growth kinetics indistinguishable from that of C-strain. Removal of the C-strain glycosylation site does not affect viral replication in rabbits and the attenuated phenotype in pigs. However, rShimen-A988T was attenuated and protected the pigs from a lethal challenge at 14 days postinoculation. In contrast, the rHCLV-T988A-inoculated pigs showed transient fever, a few clinical signs, and pathological changes in the spleens upon challenge with the Shimen strain. Mechanistic investigations revealed that the unique glycosylation at position 986 influences viral spreading, alters the formation of E2 homodimers, and leads to increased production of neutralizing antibodies. Collectively, our data for the first time demonstrate that the unique glycosylation at position 986 on the E2 glycoprotein is responsible for viral attenuation and protection. IMPORTANCE Viral glycoproteins involve in infectivity, virulence, and host immune responses. Deglycosylation on the Erns, E1, or E2 glycoprotein of highly virulent classical swine fever virus (CSFV) attenuated viral virulence in pigs, indicating that the glycosylation contributes to the pathogenicity of the highly virulent strain. However, the effects of the glycosylation on the C-strain E2 glycoprotein on viral infectivity in cells, viral attenuation, and protection in pigs have not been elucidated. This study demonstrates the unique glycosylation at position 986 on the C-strain E2 glycoprotein. C-strain mutant removing the glycosylation at the site provides only partial protection against CSFV challenge. Remarkably, the addition of the glycan to E2 of the highly virulent Shimen strain attenuates the viral virulence and confers complete protection against the lethal challenge in pigs. Our findings provide a new insight into the contribution of the glycosylation to the virus attenuation and protection.
Asunto(s)
Virus de la Fiebre Porcina Clásica/inmunología , Virus de la Fiebre Porcina Clásica/patogenicidad , Peste Porcina Clásica/prevención & control , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/metabolismo , Glicosilación , Inmunización/veterinaria , Mutación , Multimerización de Proteína , Conejos , Porcinos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/metabolismo , Virulencia , Replicación ViralRESUMEN
The sera from pigs infected with virulent classical swine fever virus (CSFV) contain substantial amounts of tumor necrosis factor (TNF), a prototype proinflammatory cytokine with pleiotropic activities. TNF limits the replication of CSFV in cell culture. In order to investigate the signaling involved in the antiviral activity of TNF, we employed small-molecule inhibitors to interfere specifically with JAK/STAT and NF-κB signaling pathways in near-to-primary endothelial PEDSV.15 cells. In addition, we knocked out selected factors of the interferon (IFN) induction and signaling pathways using CRISPR/Cas9. We found that the anti-CSFV effect of TNF was sensitive to JAK/STAT inhibitors, suggesting that TNF induces IFN signaling. Accordingly, we observed that the antiviral effect of TNF was dependent on intact type I IFN signaling as PEDSV.15 cells with the disrupted type I IFN receptor lost their capacity to limit the replication of CSFV after TNF treatment. Consequently, we examined whether TNF activates the type I IFN induction pathway. With genetically modified PEDSV.15 cells deficient in functional interferon regulatory factor 1 or 3 (IRF1 or IRF3), we observed that the anti-CSFV activity exhibited by TNF was dependent on IRF1, whereas IRF3 was dispensable. This was distinct from the lipopolysaccharide (LPS)-driven antiviral effect that relied on both IRF1 and IRF3. In agreement with the requirement of IRF1 to induce TNF- and LPS-mediated antiviral effects, intact IRF1 was also essential for TNF- and LPS-mediated induction of IFN-ß mRNA, while the activation of NF-κB was not dependent on IRF1. Nevertheless, NF-κB activation was essential for the TNF-mediated antiviral effect. Finally, we observed that CSFV failed to counteract the TNF-mediated induction of the IFN-ß mRNA in PEDSV.15 cells, suggesting that CSFV does not interfere with IRF1-dependent signaling. In summary, we report that the proinflammatory cytokine TNF limits the replication of CSFV in PEDSV.15 cells by specific induction of an IRF1-dependent antiviral type I IFN response.
Asunto(s)
Virus de la Fiebre Porcina Clásica/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/fisiología , Animales , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/efectos de los fármacos , Virus de la Fiebre Porcina Clásica/patogenicidad , Citocinas/metabolismo , Expresión Génica/genética , Regulación Viral de la Expresión Génica/genética , Interacciones Huésped-Patógeno , Factor 1 Regulador del Interferón/metabolismo , Interferón beta/genética , Interferones/metabolismo , Janus Quinasa 1/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Porcinos , Inhibidores del Factor de Necrosis Tumoral/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The classical swine fever virus (CSFV) glycoprotein E2 is the major structural component of the virus particle. E2 is involved in several functions, such as virus adsorption to the cell, the elicitation of protective immune responses, and virus virulence in swine. Using a yeast two-hybrid system, we previously identified the swine host protein Torsin-1A, an ATPase protein residing in the endoplasmic reticulum and inner nucleus membrane of the cell, as a specific binding partner for E2. The interaction between Torsin-1A and E2 proteins was confirmed to occur in CSFV-infected swine cells using three independent methods: coimmunoprecipitation, confocal microscopy, and proximity ligation assay (PLA). Furthermore, the E2 residue critical to mediate the protein-protein interaction with Torsin-1A was identified by a reverse yeast two-hybrid assay using a randomly mutated E2 library. A recombinant CSFV E2 mutant protein with a Q316L substitution failed to bind swine Torsin-1A in the yeast two-hybrid model. In addition, a CSFV infectious clone harboring the E2 Q316L substitution, although expressing substantial levels of E2 protein, repetitively failed to produce virus progeny when the corresponding RNA was transfected into susceptible SK6 cells. Importantly, PLA analysis of the transfected cells demonstrated an abolishment of the interaction between E2 Q316L and Torsin-1A, indicating a critical role for that interaction during CSFV replication.IMPORTANCE Structural glycoprotein E2 is an important structural component of the CSFV particle. E2 is involved in several virus functions, particularly virus-host interactions. Here, we characterized the interaction between CSFV E2 and swine protein Torsin-1A during virus infection. The critical amino acid residue in E2 mediating the interaction with Torsin-1A was identified and the effect of disrupting the E2-Torsin-1A protein-protein interaction was studied using reverse genetics. It is shown that the amino acid substitution abrogating E2-Torsin-1A interaction constitutes a lethal mutation, demonstrating that this virus-host protein-protein interaction is a critical factor during CSFV replication. This highlights the potential importance of the E2-Torsin-1A protein-protein interaction during CSFV replication and provides a potential pathway toward blocking virus replication, an important step toward the potential development of novel virus countermeasures.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Chaperonas Moleculares/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular , Virus de la Fiebre Porcina Clásica/metabolismo , Interacciones Huésped-Patógeno , Chaperonas Moleculares/genética , Mutación , Unión Proteica , Proteínas Recombinantes/metabolismo , Porcinos , Técnicas del Sistema de Dos Híbridos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Replicación ViralRESUMEN
Classical swine fever virus (CSFV), an enveloped virus belonging to the genus Pestivirus of the Flaviviridae family, utilizes cell host factors for its own replication. ARFGAP1, GTPase activating protein of ADP-ribosylation factor 1, regulates COP I vesicle formation and function in cells and is involved in the life cycle of several viruses. However, the effect of ARFGAP1 on the infection of CSFV has not been illustrated. Here we showed that inhibition of ARFGAP1 either by QS11 or by lentivirus-mediated silencing repressed CSFV replication. While, subsequent experiments revealed that CSFV production were increased in cells with sufficient ARFGAP1 expression. However, ARFGAP1 was not involved in CSFV binding, entry, access to cell vesicles, and RNA replication during the early stages of infection. Then, we showed that ARFGAP1 interacted with the viral protein of NS5A, measured by immunoprecipitation, GST-pulldown, and confocal microscopy assays. Furthermore, we revealed that ARFGAP1 could alleviated CSFV NS5A-induced endoplasmic reticulum stress (ERS). Altogether, these results demonstrate that ARFGAP1, a NS5A binding protein, is involved in CSFV replication.
Asunto(s)
Virus de la Fiebre Porcina Clásica/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Animales , Línea Celular , Supervivencia Celular , Estrés del Retículo Endoplásmico , Proteínas Activadoras de GTPasa/genética , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Unión Proteica , Purinas/farmacología , Porcinos , Proteínas Virales/genéticaRESUMEN
Classical swine fever virus (CSFV) is an important pathogen in the swine industry. Virion attachment is mediated by envelope proteins Erns and E2, and E2 is indispensable. Using a pull-down assay with soluble E2 as the bait, we demonstrated that ADAM17, a disintegrin and metalloproteinase 17, is essential for CSFV entry. Loss of ADAM17 in a permissive cell line eliminated E2 binding and viral entry, but compensation with pig ADAM17 cDNA completely rescued these phenotypes. Similarly, ADAM17 silencing in primary porcine fibroblasts significantly impaired virus infection. In addition, human and mouse ADAM17, which is highly homologous to pig ADAM17, also mediated CSFV entry. The metalloproteinase domain of ADAM17 bound directly to E2 protein in a zinc-dependent manner. A surface exposed region within this domain was mapped and shown to be critical for CSFV entry. These findings clearly demonstrate that ADAM17 serves as an essential attachment factor for CSFV.
Asunto(s)
Proteína ADAM17/metabolismo , Virus de la Fiebre Porcina Clásica/metabolismo , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Animales , Peste Porcina Clásica , Virus de la Fiebre Porcina Clásica/patogenicidad , Humanos , PorcinosRESUMEN
Interactions between the major structural glycoprotein E2 of classical swine fever virus (CSFV) with host proteins have been identified as important factors affecting virus replication and virulence. Previously, using the yeast two-hybrid system, we identified swine host proteins specifically interacting with CSFV E2. In this report, we use a proximity ligation assay to demonstrate that swine host protein CCDC115 interacts with E2 in CSFV-infected swine cells. Using a randomly mutated E2 library in the context of a yeast two-hybrid methodology, specific amino acid mutations in the CSFV E2 protein responsible for disrupting the interaction with CCDC115 were identified. A recombinant CSFV mutant (E2ΔCCDC115v) harboring amino acid changes disrupting the E2 protein interaction with CCDC115 was produced and used as a tool to assess the role of the E2-CCDC115 interaction in viral replication and virulence in swine. CSFV E2ΔCCDC115v showed a slightly decreased ability to replicate in the SK6 swine cell line and a greater replication defect in primary swine macrophage cultures. A decreased E2-CCDC115 interaction detected by PLA is observed in cells infected with E2ΔCCDC115v. Importantly, animals intranasally infected with 105 TCID50 of E2ΔCCDC115v experienced a significantly longer survival period when compared with those infected with the parental Brescia strain. This result would indicate that the ability of CSFV E2 to bind host CCDC115 protein during infection plays an important role in virus replication in swine macrophages and in virus virulence during the infection in domestic swine.
Asunto(s)
Virus de la Fiebre Porcina Clásica/fisiología , Peste Porcina Clásica/virología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Replicación Viral , Animales , Células Cultivadas , Peste Porcina Clásica/metabolismo , Peste Porcina Clásica/patología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/metabolismo , Virus de la Fiebre Porcina Clásica/patogenicidad , Interacciones Huésped-Patógeno , Macrófagos/virología , Mutación , Unión Proteica , Análisis de Supervivencia , Porcinos , Proteínas del Envoltorio Viral/genética , Virulencia/genética , Replicación Viral/genéticaRESUMEN
The E2 envelope protein is the main protective antigen of classical swine fever virus (CSFV). Importantly, gram-positive enhancer matrix (GEM) particles can work as an immunostimulant and/or carrier system to improve the immune effect of antigens. In this study, the artificially designed E2-Spy was expressed and glycosylated in Pichia pastoris, and subsequently conjugated with SpyCatcher-PA which was expressed in Escherichia coli. The conjugated E2-Spy-PA was displayed on the surface of GEM particles, generating the E2-Spy-PA-GEM complex. Blocking ELISA analysis and neutralization assays showed that both E2-Spy and E2-Spy-PA-GEM complexes induced high levels of anti-CSFV antibodies in mice. Furthermore, statistical analyses indicated that the E2-Spy-PA-GEM complex exhibited enhanced immunogenicity compared with E2-Spy alone.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus de la Fiebre Porcina Clásica/inmunología , Proteínas del Envoltorio Viral , Inmunidad Adaptativa , Animales , Virus de la Fiebre Porcina Clásica/metabolismo , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Ratones , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Saccharomycetales/genética , Porcinos , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.
Asunto(s)
Clonación Molecular/métodos , Proteínas del Envoltorio Viral , Animales , Virus de la Fiebre Porcina Clásica/metabolismo , Ratones , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Saccharomycetales/genética , Porcinos , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genéticaRESUMEN
Classical swine fever virus (CSFV) causes a contagious disease of pigs. The virus can break the mucosal barrier to establish its infection. Type III interferons (IFN-λs) play a crucial role in maintaining the antiviral state in epithelial cells. Limited information is available on whether or how CSFV modulates IFN-λs production. We found that IFN-λ3 showed dose-dependent suppression of CSFV replication in IPEC-J2 cells. Npro-deleted CSFV mutant (∆Npro) induced significantly higher IFN-λs transcription from 24 h post-infection (hpi) than its parental strain (wtCSFV). The strain wtCSFV strongly inhibited IFN-λs transcription and IFN-λ3 promoter activity in poly(I:C)-stimulated IPEC-J2 cells, whereas ∆Npro did not show such inhibition. Npro overexpression caused significant reduction of IFN-λs transcription and IFN-λ3 promoter activity. Both wtCSFV and ∆Npro infection induced time-dependent IRF1 expression in IPEC-J2 cells, with ΔNpro showing more significant induction, particularly at 24 hpi. However, infection with wtCSFV or Npro overexpression led not only to significant reduction of IRF1 expression and its promoter activity in poly(I:C)-treated IPEC-J2 cells but also to blockage of IRF1 nuclear translocation. This study provides clear evidence that CSFV Npro suppresses IRF1-mediated type III IFNs production by inhibiting IRF1 expression and its nuclear translocation.
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Virus de la Fiebre Porcina Clásica , Peste Porcina Clásica/inmunología , Endopeptidasas , Interferones/metabolismo , Proteínas Virales , Transporte Activo de Núcleo Celular/inmunología , Animales , Línea Celular , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Inmunidad Innata , Factor 1 Regulador del Interferón/metabolismo , Mutación/genética , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Interferón lambdaRESUMEN
BACKGROUND: Capsid (C) protein plays an important role in the replication of classical swine fever virus (CSFV). The ubiquitin proteasome system (UPS) involves in replication of many viruses via modulation of viral proteins. The relationship of CSFV with UPS is poorly understood and the impact of 26S proteasome on C protein has never been reported before. METHODS: In this study, fused C protein with an EGFP tag is expressed in PK-15 and 3D4/2 cells. MG132 and 3-methyladenine (3-MA) are used to detect the roles of 26S proteasome and autophagolysosome in expression levels of C protein. Truncated and mutant C proteins are used to find the exact residues responsible for the degradation of C protein. Immunoprecipitaion is performed to find whether C protein is ubiquitinated or not. RESULTS: C-EGFP protein expresses in a cleaved form at a low level and is degraded by 26S proteasome which could be partly inhibited by MG132. C-terminal residues play more important roles in the degradation of C protein than N-terminal residues. Residues 260 to 267, especially M260 and L261, are crucial for the degradation. In addition, C-terminal residues 262 to 267 determine cleavage efficiency of C protein. CONCLUSIONS: CSFV C protein is degraded by 26S proteasome in a ubiquitin-independent manner. Last 8 residues at C-terminus of immature C protein play a major role in proteasomal degradation of CSFV C protein and determine the cleavage efficiency of C protein by signal peptide peptidase (SPP). Our findings provide valuable help for fully understanding degradation process of C protein and contribute to fully understanding the role of C protein in CSFV replication.
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Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Virus de la Fiebre Porcina Clásica/metabolismo , Aminoácidos , Animales , Proteínas de la Cápside/genética , Línea Celular , Peste Porcina Clásica/virología , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , PorcinosRESUMEN
The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence.IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.
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Virus de la Fiebre Porcina Clásica/genética , Peste Porcina Clásica/virología , Complejo Dinactina/genética , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Proteínas del Envoltorio Viral/genética , Animales , Sitios de Unión , Línea Celular , Peste Porcina Clásica/mortalidad , Peste Porcina Clásica/patología , Virus de la Fiebre Porcina Clásica/metabolismo , Virus de la Fiebre Porcina Clásica/patogenicidad , Complejo Dinactina/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/virología , Biblioteca de Genes , Macrófagos/metabolismo , Macrófagos/virología , Mutación , Cultivo Primario de Células , Unión Proteica , Transducción de Señal , Análisis de Supervivencia , Porcinos , Técnicas del Sistema de Dos Híbridos , Proteínas del Envoltorio Viral/metabolismo , Replicación ViralRESUMEN
For members of the Flaviviridae, it is known that, besides the structural proteins, nonstructural (NS) proteins also play a critical role in virion formation. Pestiviruses, such as bovine viral diarrhea virus (BVDV), rely on uncleaved NS2-3 for virion formation, while its cleavage product, NS3, is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in the absence of uncleaved NS2-3 in BVDV type 1 (BVDV-1) variants encoding either a ubiquitin (Ubi) (NS2-Ubi-NS3) or an internal ribosome entry site (IRES) (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were capable of restoring low-level NS2-3-independent virion formation only for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D), identified by selection, was essential for high-titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed for low-titer virion production only in CSFV NS2-IRES-NS3. For efficient virion morphogenesis, additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RNA-dependent RNA polymerase, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on the genome packaging of pestiviruses.IMPORTANCE For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.
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Virus de la Fiebre Porcina Clásica/fisiología , Cisteína Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/metabolismo , Cisteína Endopeptidasas/genética , Pestivirus/genética , Pestivirus/metabolismo , ARN Helicasas/metabolismo , ARN Viral/genética , Porcinos , Virión/genética , Virión/metabolismo , Ensamble de Virus , Replicación ViralRESUMEN
Classical swine fever (CSF) is a highly contagious swine disease that causes devastating economic losses. However, there are few efficacious therapeutic antibodies against the CSF virus (CSFV). Accordingly, we isolated two whole-porcine anti-CSFV neutralizing antibodies (NAbs) directly from single B cells sorted using the conserved linear epitope of the CSFV E2 protein and goat anti-pig IgG. These mAbs, termed HK24 and HK44, can bind to the E2 protein by recognizing sites within the conserved linear epitope of E2. In addition, these two mAbs can detect virus infection with high specificity and possess potent neutralizing activity. HK24 and HK44 protect PK-15 cells from CSFV infections in vitro with potent IC50 values of 9.3 and 0.62 µg/mL, respectively. We anticipate that these antibodies can be used as diagnostic and antiviral agents for CSFV and that the method we describe here will accelerate the production of therapeutic antibodies for other viruses.
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Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/genética , Linfocitos B/metabolismo , Virus de la Fiebre Porcina Clásica/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/metabolismo , Reacciones Antígeno-Anticuerpo , Linfocitos B/citología , Línea Celular , Virus de la Fiebre Porcina Clásica/metabolismo , Epítopos/química , Epítopos/inmunología , Epítopos/metabolismo , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Análisis de la Célula Individual , Porcinos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDVE2_fluo). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDVE2_fluo particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDVE2_fluo as a tool to study the dynamics of the whole life cycle of BVDV in real time.