Screening of cellular proteins that interact with the classical swine fever virus non-structural protein 5A by yeast two-hybrid analysis

E2 is the major structural glycoprotein of the classical swine fever virus (CSFV). E2 has been shown to be involved in important virus functions such as replication and virulence in swine. Using the yeast two-hybrid system, we previously identified several host proteins specifically interacting with CSFV E2. Here, we analyze the protein interaction of E2 with SERTA domain containing protein 1 (SERTAD1), a factor involved in the stimulation of the transcriptional activities of different host genes. We have confirmed that the interaction between these two proteins occurs in CSFV-infected swine cells by using a proximity ligation assay and confocal microscopy. Amino acid residues in the CSFV E2 protein that are responsible for mediating the interaction with SERTAD1 were mapped by a yeast two-hybrid approach using a randomly mutated E2 library. Using that information, a recombinant CSFV mutant (E2ΔSERTAD1v) that harbors substitutions in those residues mediating the protein-interaction with SERTAD1 was developed and used to study the role of the E2-SERTAD1 interaction in viral replication and virulence in swine. CSFV E2ΔSERTAD1v, when compared to the parental BICv, showed a clearly decreased ability to replicate in the SK6 swine cell line and a more severe replication defect in primary swine macrophage cultures. Importantly, 80% of animals infected with E2ΔSERTAD1v survived infection, remaining clinically normal during the 21-day observational period. This result would indicate that the ability of CSFV E2 to bind host SERTAD1 protein during infection plays a critical role in virus virulence.

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The Npro product of classical swine fever virus interacts with IκBα, the NF-κB inhibitor

Journal of General Virology ◽

10.1099/vir.0.83643-0 ◽

2008 ◽

Vol 89 (8) ◽

pp. 1881-1889 ◽

Cited By ~ 43

Author(s):

Virginie Doceul ◽

Bryan Charleston ◽

Helen Crooke ◽

Elizabeth Reid ◽

Penny P. Powell ◽

...

Keyword(s):

Immune Response ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Host Cells ◽

Nuclear Accumulation ◽

Yeast Two Hybrid ◽

Necrosis Factor Alpha ◽

Induced Apoptosis ◽

Two Hybrid

Classical swine fever virus (CSFV) belongs to the genus Pestivirus and is the causative agent of classical swine fever, a haemorrhagic disease of pigs. The virus replicates in host cells without activating interferon (IFN) production and has been reported to be an antagonist of double-stranded RNA-induced apoptosis. The N-terminal protease (Npro) of CSFV is responsible for this evasion of the host innate immune response. In order to identify cellular proteins that interact with the Npro product of CSFV, a yeast two-hybrid screen of a human library was carried out, which identified IκBα, the inhibitor of NF-κB, a transcription factor involved in the control of apoptosis, the immune response and IFN production. The Npro–IκBα interaction was confirmed using yeast two-hybrid analysis and additional co-precipitation assays. It was also shown that Npro localizes to both the cytoplasmic and nuclear compartments in stably transfected cells and in CSFV-infected cells. Following stimulation by tumour necrosis factor alpha, PK-15 cell lines expressing Npro exhibited transient nuclear accumulation of pIκBα, but no effect of CSFV infection on IκBα localization or NF-κB p65 activation was observed.

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Swine Host Protein Coiled-Coil Domain-Containing 115 (CCDC115) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2 during Virus Replication

Viruses ◽

10.3390/v12040388 ◽

2020 ◽

Vol 12 (4) ◽

pp. 388 ◽

Cited By ~ 1

Author(s):

Elizabeth A. Vuono ◽

Elizabeth Ramirez-Medina ◽

Keith Berggren ◽

Ayushi Rai ◽

Sarah Pruitt ◽

...

Keyword(s):

Amino Acid ◽

Virus Replication ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Host Protein ◽

Yeast Two Hybrid ◽

Host Proteins ◽

E2 Protein ◽

Two Hybrid

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.

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Identification of two internal signal peptide sequences: critical for classical swine fever virus non-structural protein 2 to trans-localize to the endoplasmic reticulum

Virology Journal ◽

10.1186/1743-422x-8-236 ◽

2011 ◽

Vol 8 (1) ◽

pp. 236 ◽

Cited By ~ 8

Author(s):

Kang-kang Guo ◽

Qing-hai Tang ◽

Yan-ming Zhang ◽

Kai Kang ◽

Lei He

Keyword(s):

Endoplasmic Reticulum ◽

Signal Peptide ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Structural Protein ◽

Internal Signal

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Identification of T-cell epitopes in the structural and non-structural proteins of classical swine fever virus

Journal of General Virology ◽

10.1099/0022-1317-83-3-551 ◽

2002 ◽

Vol 83 (3) ◽

pp. 551-560 ◽

Cited By ~ 53

Author(s):

Elisenda Armengol ◽

Karl-Heinz Wiesmüller ◽

Daniel Wienhold ◽

Mathias Büttner ◽

Eberhard Pfaff ◽

...

Keyword(s):

T Cell ◽

Mhc Class Ii ◽

Classical Swine Fever Virus ◽

Cell Epitope ◽

Classical Swine Fever ◽

Fever Virus ◽

Target Cells ◽

T Cell Epitopes ◽

Structural Protein

To identify new T-cell epitopes of classical swine fever virus (CSFV), 573 overlapping, synthetic pentadecapeptides spanning 82% of the CSFV (strain Glentorf) genome sequence were synthesized and screened. In proliferation assays, 26 peptides distributed throughout the CSFV viral protein sequences were able to induce specific T-cell responses in PBMCs from a CSFV-Glentorf-infected d/d haplotype pig. Of these 26 peptides, 18 were also recognized by PBMCs from a CSFV-Alfort/187-infected d/d haplotype pig. In further experiments, it could be shown that peptide 290 (KHKVRNEVMVHWFDD), which corresponds to amino acid residues 1446–1460 of the CSFV non-structural protein NS2–3 could induce interferon-γ secretion after secondary in vitro restimulation. The major histocompatibility complex (MHC) restriction for stimulation of T-cells by this pentadecapeptide was identified as being mainly MHC class II and partially MHC class I. In cytolytic assays, CSFV-specific cytotoxic T-lymphocytes (CTLs) were able to lyse peptide 290-loaded target cells. These findings indicate the existence of a CSFV-specific helper T-cell epitope and a CTL epitope in this peptide.

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Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts

Colloids and Surfaces B Biointerfaces ◽

10.1016/j.colsurfb.2006.10.042 ◽

2007 ◽

Vol 55 (1) ◽

pp. 26-30 ◽

Cited By ~ 51

Author(s):

Dong-Mei He ◽

Kai-Xian Qian ◽

Gui-Fang Shen ◽

Zhi-Fang Zhang ◽

Yi-Nü LI ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Structural Protein ◽

E2 Gene

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Classical Swine Fever Virus ErnsDeletion Mutants: trans-Complementation and Potential Use as Nontransmissible, Modified, Live-Attenuated Marker Vaccines

Journal of Virology ◽

10.1128/jvi.74.7.2973-2980.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 2973-2980 ◽

Cited By ~ 68

Author(s):

M. N. Widjojoatmodjo ◽

H. G. P. van Gennip ◽

A. Bouma ◽

P. A. van Rijn ◽

R. J. M. Moormann

Keyword(s):

Amino Acids ◽

Cell Line ◽

Classical Swine Fever Virus ◽

Lethal Dose ◽

Classical Swine Fever ◽

Fever Virus ◽

Wild Type Virus ◽

Deletion Mutants ◽

Structural Protein ◽

Viral Particles

ABSTRACT An SK6 cell line (SK6c26) which constitutively expressed the glycoprotein Erns of classical swine fever virus (CSFV) was used to rescue CSFV Erns deletion mutants based on the infectious copy of CSFV strain C. The biochemical properties of Erns from this cell line were indistinguishable from those of CSFV Erns. Two Erns deletion mutants were constructed, virus Flc23 and virus Flc22. Virus Flc23 encoded only the utmost N- and C-terminal amino acids of Erns (deletion of 215 amino acids) to retain the original protease cleavage sites. Virus Flc22 is not recognized by a panel of Erns antibodies, due to a deletion of 66 amino acids in Erns. The Erns deletion mutants Flc22 and Flc23 could be rescued in vitro only on the complementing SK6c26 cells. These rescued viruses could infect and replicate in SK6 cells but did not yield infectious virus. Virus neutralization by Erns-specific antibodies was similar for the wild-type virus and the recombinant viruses, indicating that Erns from SK6c26 cells was incorporated in the viral particles. Pigs vaccinated with Flc22 or Flc23 were protected against a challenge with a lethal dose of CSFV strain Brescia. This is the first demonstration of trans-complementation of defective pestivirus RNA with a pestiviral structural protein and opens new ways to develop nontransmissible modified live pestivirus vaccines. In addition, the absence of (the antigenic part of) Erns in the recombinant viral particles can be used to differentiate between infected and vaccinated animals.

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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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