Npro of Classical Swine Fever Virus Suppresses Type III Interferon Production by Inhibiting IRF1 Expression and Its Nuclear Translocation

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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Classical swine fever virus NS5A inhibits NF-κB signaling by targeting NEMO

10.1101/2021.06.21.449351 ◽

2021 ◽

Author(s):

Jiaying Li ◽

Haixiao Yu ◽

Wenjin Jiang ◽

Ping Ma ◽

Zezhong Feng ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Protein Translation ◽

Proteasomal Degradation ◽

Fever Virus ◽

Poly I:C ◽

Regulatory Subunit ◽

Structural Protein ◽

Multifunctional Protein ◽

Iκb Kinase

The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation and regulation of cellular signaling pathways, and assembly of infectious virus particles. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism was not elucidated. Here, we report that NS5A interacts with NF-κB essential modulator (NEMO), a regulatory subunit of the IκB kinase (IKK) complex, and that the zinc finger domain of NEMO essential for NEMO ubiquitination and IKK activation is required for the interaction of NEMO with NS5A. Viral infection or NS5A expression by itself reduced the protein level of NEMO. Mechanistic analysis revealed that NS5A mediated proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced K27- but not K48-linked polyubiquitination of NEMO. In addition, NS5A blocked k63-linked polyubiquitination of NEMO, thereby inhibiting activation of IKK and NF-κB. These findings indicate that NS5A inhibits NF-κB signaling by mediating proteasomal degradation of NEMO and blocking k63-linked polyubiquitination of NEMO, thereby revealing a novel mechanism by which CSFV inhibits host innate immunity.

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Infection with Classical Swine Fever Virus Induces Expression of Type III Interferons and Activates Innate Immune Signaling

Frontiers in Microbiology ◽

10.3389/fmicb.2017.02558 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 10

Author(s):

Binxiang Cai ◽

Qingling Bai ◽

Xiaojuan Chi ◽

Mohsan U. Goraya ◽

Long Wang ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Innate Immune ◽

Fever Virus ◽

Immune Signaling ◽

Type Iii ◽

Innate Immune Signaling

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Classical swine fever virus Npro antagonises IRF3 to prevent IFN-independent TLR3 and RIG-I-mediated apoptosis

Journal of Virology ◽

10.1128/jvi.01136-20 ◽

2020 ◽

pp. JVI.01136-20

Author(s):

Samuel Hardy ◽

Ben Jackson ◽

Stephen Goodbourn ◽

Julian Seago

Keyword(s):

Wild Boar ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Economic Importance ◽

Fever Virus ◽

Poly I:C ◽

Type I ◽

Type I Ifn ◽

Notifiable Disease ◽

Haemorrhagic Disease

Classical swine fever virus (CSFV) is the causative agent of classical swine fever, a notifiable disease of economic importance that causes severe leukopenia, fever and haemorrhagic disease in domesticated pigs and wild boar across the globe. CSFV has been shown to antagonise the induction of type I IFN, partly through a function of its N-terminal protease (Npro) which binds IRF3 and targets it for proteasomal degradation. Additionally, Npro has been shown to antagonise apoptosis triggered by the dsRNA-homolog poly(I:C), however the exact mechanism by which this is achieved has not been fully elucidated. In this study we confirm the ability of Npro to inhibit dsRNA-mediated apoptosis and show that Npro is also able to antagonise Sendai virus-mediated apoptosis in PK-15 cells. Gene edited PK-15 cell lines were used to show the dsRNA-sensing pathogen recognition receptors (PRRs) TLR3 and RIG-I specifically respond to poly(I:C) and SeV respectively, subsequently triggering apoptosis through pathways that converge on IRF3 and culminate in the cleavage of caspase-3. Importantly, this IRF3-mediated apoptosis was found to be dependent on transcription-independent functions of IRF3 and also on Bax, a pro-apoptotic Bcl-2 family protein, through a direct interaction between the two proteins. Deletion of IRF3, stable expression of Npro and infection with wild-type CSFV were found to antagonise the mitochondrial localisation of Bax, a key hallmark of the intrinsic, mitochondrial pathway of apoptosis. Together, these findings show that Npro’s putative interaction with IRF3 is involved not only in its antagonism of type I IFN, but also dsRNA-mediated mitochondrial apoptosis.Importance Responsible for severe haemorrhagic disease in domestic pigs and wild boar, classical swine fever is recognised by the World Organisation for Animal Health (OIE) and European Union as a notifiable disease of economic importance. Persistent infection, immunotolerance and early dissemination of the virus at local sites of infection have been linked to the antagonism of type I IFN induction by Npro. This protein may further contribute to these phenomena by antagonising the induction of dsRNA-mediated apoptosis. Ultimately, apoptosis is an important innate mechanism by which cells counter viruses at local sites of infection, thus preventing wider spread and dissemination within the host, potentially also contributing to the onset of persistence. Elucidation of the mechanism by which Npro antagonises the apoptotic response will help inform the development of rationally-designed live-attenuated vaccines and antivirals for control of outbreaks in typically CSFV-free countries.

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Thioredoxin 2 Is a Novel E2-Interacting Protein That Inhibits the Replication of Classical Swine Fever Virus

Journal of Virology ◽

10.1128/jvi.00429-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8510-8524 ◽

Cited By ~ 21

Author(s):

Su Li ◽

Jinghan Wang ◽

Wen-Rui He ◽

Shuo Feng ◽

Yongfeng Li ◽

...

Keyword(s):

Viral Replication ◽

Signaling Pathway ◽

Classical Swine Fever Virus ◽

Nuclear Translocation ◽

Classical Swine Fever ◽

Fever Virus ◽

E2 Protein ◽

Cellular Events ◽

Thioredoxin 2

ABSTRACTThe E2 protein of classical swine fever virus (CSFV) is an envelope glycoprotein that is involved in virus attachment and entry. To date, the E2-interacting cellular proteins and their involvement in viral replication have been poorly documented. In this study, thioredoxin 2 (Trx2) was identified to be a novel E2-interacting partner using yeast two-hybrid screening from a porcine macrophage cDNA library. Trx2 is a mitochondrion-associated protein that participates in diverse cellular events. The Trx2-E2 interaction was further confirmed by glutathioneS-transferase (GST) pulldown,in situproximity ligation, and laser confocal assays. The thioredoxin domain of Trx2 and the asparagine at position 37 (N37) in the E2 protein were shown to be critical for the interaction. Silencing of the Trx2 expression in PK-15 cells by small interfering RNAs significantly promotes CSFV replication, and conversely, overexpression of Trx2 markedly inhibits viral replication of the wild-type (wt) CSFV and to a greater extent that of the CSFV N37D mutant, which is defective in binding Trx2. The wt CSFV but not the CSFV N37D mutant was shown to reduce the Trx2 protein expression in PK-15 cells. Furthermore, we demonstrated that Trx2 increases nuclear factor kappa B (NF-κB) promoter activity by promoting the nuclear translocation of the p65 subunit of NF-κB. Notably, activation of the NF-κB signaling pathway induced by tumor necrosis factor alpha (TNF-α) significantly inhibits CSFV replication in PK-15 cells, whereas blocking the NF-κB activation in Trx2-overexpressing cells no longer suppresses CSFV replication. Taken together, our findings reveal that Trx2 inhibits CSFV replication via the NF-κB signaling pathway.IMPORTANCEThioredoxin 2 (Trx2) is a mitochondrion-associated protein that participates in diverse cellular events, such as antioxidative and antiapoptotic processes and the modulation of transcription factors. However, little is known about the involvement of Trx2 in viral replication. Here, we investigated, for the first time, the role of Trx2 in the replication of classical swine fever virus (CSFV), a devastating pestivirus of pigs. By knockdown and overexpression, we showed that Trx2 negatively regulates CSFV replication. Notably, we demonstrated that Trx2 inhibits CSFV replication by promoting the nuclear translocation of the p65 subunit of NF-κB, a key regulator of the host's innate immunity and inflammatory response. Our findings reveal a novel role of Trx2 in the host's antiviral response and provide new insights into the complex mechanisms by which CSFV interacts with the host cell.

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ARFGAP1 binds to classical swine fever virus NS5A protein and enhances CSFV replication in PK-15 cells

Veterinary Microbiology ◽

10.1016/j.vetmic.2021.109034 ◽

2021 ◽

Vol 255 ◽

pp. 109034

Author(s):

Liang Zhang ◽

Mingxing Jin ◽

Mengzhao Song ◽

Shanchuan Liu ◽

Tao Wang ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Ns5a Protein

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Genome Variability of Classical Swine Fever Virus during the 2018–2020 Epidemic in Japan

Veterinary Microbiology ◽

10.1016/j.vetmic.2021.109128 ◽

2021 ◽

pp. 109128

Author(s):

Tatsuya Nishi ◽

Katsuhiko Fukai ◽

Tomoko Kato ◽

Kotaro Sawai ◽

Takehisa Yamamoto

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Genome Variability

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A Novel E2 Glycoprotein Subunit Marker Vaccine Produced in Plant Is Able to Prevent Classical Swine Fever Virus Vertical Transmission after Double Vaccination

Vaccines ◽

10.3390/vaccines9050418 ◽

2021 ◽

Vol 9 (5) ◽

pp. 418

Author(s):

Youngmin Park ◽

Yeonsu Oh ◽

Miaomiao Wang ◽

Llilianne Ganges ◽

José Alejandro Bohórquez ◽

...

Keyword(s):

Vertical Transmission ◽

Classical Swine Fever Virus ◽

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Vaccine Dose ◽

Classical Swine Fever ◽

Fever Virus ◽

Tissue Samples ◽

Marker Vaccine ◽

E2 Glycoprotein

The efficacy of a novel subunit vaccine candidate, based in the CSFV E2 glycoprotein produced in plants to prevent classical swine fever virus (CSFV) vertical transmission, was evaluated. A Nicotiana benthamiana tissue culture system was used to obtain a stable production of the E2-glycoprotein fused to the porcine Fc region of IgG. Ten pregnant sows were divided into three groups: Groups 1 and 2 (four sows each) were vaccinated with either 100 μg/dose or 300 μg/dose of the subunit vaccine at 64 days of pregnancy. Group 3 (two sows) was injected with PBS. Groups 1 and 2 were boosted with the same vaccine dose. At 10 days post second vaccination, the sows in Groups 2 and 3 were challenged with a highly virulent CSFV strain. The vaccinated sows remained clinically healthy and seroconverted rapidly, showing efficient neutralizing antibodies. The fetuses from vaccinated sows did not show gross lesions, and all analyzed tissue samples tested negative for CSFV replication. However, fetuses of non-vaccinated sows had high CSFV replication in tested tissue samples. The results suggested that in vaccinated sows, the plant produced E2 marker vaccine induced the protective immunogenicity at challenge, leading to protection from vertical transmission to fetuses.

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