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

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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Structural glycoprotein E2 of classical swine fever virus critically interacts with host protein Torsin-1A during the virus infectious cycle

Journal of Virology ◽

10.1128/jvi.00314-21 ◽

2021 ◽

Author(s):

E. A. Vuono ◽

E. Ramirez-Medina ◽

L. Velazquez-Salinas ◽

K. Berggren ◽

A. Rai ◽

...

Keyword(s):

Amino Acid ◽

Protein Interaction ◽

Classical Swine Fever Virus ◽

Structural Component ◽

Classical Swine Fever ◽

Host Protein ◽

Yeast Two Hybrid ◽

Protein Protein Interaction ◽

Glycoprotein E2 ◽

Two Hybrid

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 methodologies: co-immunoprecipitation, 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 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 characterize 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 towards blocking virus replication, an important step towards the potential development of novel virus countermeasures.

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Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

Journal of Virology ◽

10.1128/jvi.01642-19 ◽

2019 ◽

Vol 94 (1) ◽

Cited By ~ 3

Author(s):

M. V. Borca ◽

E. A. Vuono ◽

E. Ramirez-Medina ◽

P. Azzinaro ◽

K. A. Berggren ◽

...

Keyword(s):

Amino Acid ◽

Protein Interaction ◽

Hybrid Approach ◽

Classical Swine Fever ◽

Host Protein ◽

Yeast Two Hybrid ◽

Protein Protein Interaction ◽

Viral Virulence ◽

Glycoprotein E2 ◽

Two Hybrid

ABSTRACT 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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SERTA Domain Containing Protein 1 (SERTAD1) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2, Which Is Involved in Virus Virulence in Swine

Viruses ◽

10.3390/v12040421 ◽

2020 ◽

Vol 12 (4) ◽

pp. 421

Author(s):

Elizabeth A. Vuono ◽

Elizabeth Ramirez-Medina ◽

Paul Azzinaro ◽

Keith A. Berggren ◽

Ayushi Rai ◽

...

Keyword(s):

Protein Interaction ◽

Classical Swine Fever Virus ◽

Critical Role ◽

Hybrid Approach ◽

Classical Swine Fever ◽

Fever Virus ◽

Proximity Ligation Assay ◽

Yeast Two Hybrid ◽

Clinically Normal ◽

Two Hybrid

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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Interactive cellular proteins related to classical swine fever virus non-structure protein 2 by yeast two-hybrid analysis

Molecular Biology Reports ◽

10.1007/s11033-012-1936-x ◽

2012 ◽

Vol 39 (12) ◽

pp. 10515-10524 ◽

Cited By ~ 13

Author(s):

Kai Kang ◽

Kangkang Guo ◽

Qinhai Tang ◽

Yanming Zhang ◽

Jiang Wu ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Cellular Proteins ◽

Yeast Two Hybrid ◽

Hybrid Analysis ◽

Two Hybrid

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

Journal of Biosciences ◽

10.1007/s12038-013-9411-y ◽

2014 ◽

Vol 39 (1) ◽

pp. 63-74 ◽

Cited By ~ 14

Author(s):

Chengcheng Zhang ◽

Lei He ◽

Kai Kang ◽

Heng Chen ◽

Lei Xu ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fever Virus ◽

Cellular Proteins ◽

Structural Protein ◽

Yeast Two Hybrid ◽

Hybrid Analysis ◽

Two Hybrid

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Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

Virology ◽

10.1016/j.virol.2014.03.005 ◽

2014 ◽

Vol 456-457 ◽

pp. 121-130 ◽

Cited By ~ 15

Author(s):

I.J. Fernández-Sainz ◽

E. Largo ◽

D.P. Gladue ◽

P. Fletcher ◽

V. O’Donnell ◽

...

Keyword(s):

Amino Acid ◽

Virus Replication ◽

Classical Swine Fever Virus ◽

Classical Swine Fever ◽

Fusion Peptide ◽

Fever Virus ◽

Amino Acid Substitutions ◽

Specific Amino Acid ◽

Glycoprotein E2

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Immunogenicity in Swine of Orally Administered Recombinant Lactobacillus plantarum Expressing Classical Swine Fever Virus E2 Protein in Conjunction with Thymosin α-1 as an Adjuvant

Applied and Environmental Microbiology ◽

10.1128/aem.00127-15 ◽

2015 ◽

Vol 81 (11) ◽

pp. 3745-3752 ◽

Cited By ~ 27

Author(s):

Yi-Gang Xu ◽

Xue-Ting Guan ◽

Zhong-Mei Liu ◽

Chang-Yong Tian ◽

Li-Chun Cui

Keyword(s):

Immune Responses ◽

Lactobacillus Plantarum ◽

Classical Swine Fever Virus ◽

Oral Vaccine ◽

Classical Swine Fever ◽

Fever Virus ◽

Economic Losses ◽

E2 Protein ◽

Content Type ◽

Cellular Immune

ABSTRACTClassical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious disease that results in enormous economic losses in pig industries. The E2 protein is one of the main structural proteins of CSFV and is capable of inducing CSFV-neutralizing antibodies and cytotoxic T lymphocyte (CTL) activitiesin vivo. Thymosin α-1 (Tα1), an immune-modifier peptide, plays a very important role in the cellular immune response. In this study, genetically engineeredLactobacillus plantarumbacteria expressing CSFV E2 protein alone (L. plantarum/pYG-E2) and in combination with Tα1 (L. plantarum/pYG-E2-Tα1) were developed, and the immunogenicity of each as an oral vaccine to induce protective immunity against CSFV in pigs was evaluated. The results showed that recombinantL. plantarum/pYG-E2 andL. plantarum/pYG-E2-Tα1 were both able to effectively induce protective immune responses in pigs against CSFV infection by eliciting immunoglobulin A (IgA)-based mucosal, immunoglobulin G (IgG)-based humoral, and CTL-based cellular immune responses via oral vaccination. Significant differences (P< 0.05) in the levels of immune responses were observed betweenL. plantarum/pYG-E2-Tα1 andL. plantarum/pYG-E2, suggesting a better immunogenicity ofL. plantarum/pYG-E2-Tα1 as a result of the Tα1 molecular adjuvant that can enhance immune responsiveness and augment specific lymphocyte functions. Our data suggest that the recombinantLactobacillusmicroecological agent expressing CSFV E2 protein combined with Tα1 as an adjuvant provides a promising strategy for vaccine development against CSFV.

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