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

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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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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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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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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Definition of a minimal munc18c domain that interacts with syntaxin 4

Biochemical Journal ◽

10.1042/bj3500741 ◽

2000 ◽

Vol 350 (3) ◽

pp. 741-746 ◽

Cited By ~ 5

Author(s):

Julian GRUSOVIN ◽

Violet STOICHEVSKA ◽

Keith H. GOUGH ◽

Katrina NUNAN ◽

Colin W. WARD ◽

...

Keyword(s):

Protein Interaction ◽

Full Length ◽

Yeast Two Hybrid ◽

Protein Protein Interaction ◽

Interaction Studies ◽

Syntaxin 4 ◽

Definition Of ◽

Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

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Yeast Two-Hybrid Protein-Protein Interaction Networks

Proteomics and Protein-Protein Interactions ◽

10.1007/0-387-24532-4_2 ◽

2006 ◽

pp. 19-31 ◽

Cited By ~ 4

Author(s):

Daniel Auerbach ◽

Igor Stagljar

Keyword(s):

Protein Interaction ◽

Protein Interaction Networks ◽

Interaction Networks ◽

Hybrid Protein ◽

Yeast Two Hybrid ◽

Protein Protein Interaction ◽

Protein Protein Interaction Networks ◽

Two Hybrid

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The Application of Large-scale Yeast Two-hybrid to Study Protein — Protein Interaction

Hereditas (Beijing) ◽

10.1360/yc-006-1627 ◽

2006 ◽

Vol 28 (12) ◽

pp. 1627 ◽

Cited By ~ 1

Author(s):

Zhi-Hao WU

Keyword(s):

Protein Interaction ◽

Large Scale ◽

Yeast Two Hybrid ◽

Protein Protein Interaction ◽

Two Hybrid

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Different subtypes of influenza A viruses target different human proteins and pathways leading to different pathogenic phenotypes

10.1101/541177 ◽

2019 ◽

Author(s):

Yujie Wang ◽

Ting Song ◽

Kaiwu Li ◽

Yuan Jin ◽

Junjie Yue ◽

...

Keyword(s):

Influenza A ◽

Influenza Viruses ◽

Host Protein ◽

Yeast Two Hybrid ◽

Influenza A Viruses ◽

Protein Protein Interaction ◽

Ppi Networks ◽

Tnf Α ◽

Human Proteins ◽

Two Hybrid

AbstractDifferent subtypes of Influenza A viruses cause different pathogenic phenotypes after infecting human bodies. Direct binary interactions between viral proteins and human proteins provide an important background for influenza viruses to cause complex pathologies of hosts. Here, we demonstrated the different impacts on the TNF-α-induced NF-κB activation of H1N1 and H5N1 virus proteins. By further examining the virus-host protein-protein interactions (PPI), we found that the same segment protein of the H1N1 and H5N1 viruses target on different host proteins. We then performed a yeast two-hybrid analysis of a highly pathogenic avian H5N1 influenza virus and human proteins. Influenza-host protein-protein interaction networks of three strains of influenza A viruses (including two other reported influenza-host PPI networks) were systematically compared and mapped on the network level and the pathway level. The results show subtype-specific characters of the influenza-host protein interactome, which may response for the specific pathogenic mechanisms of different subtypes of influenza viruses.ImportanceInfluenza A virus (IAV) can cause contagious respiratory illness, namely influenza (flu). The symptoms of infections from different subtypes of IAVs vary from mild to severe illness. The mechanism of these different pathogenic phenotypes remains poorly understood. Our results show that the same NA and NP segments from H1N1 and H5N1 virus cause different impacts on the TNF-α-induced NF-κB pathway. Furthermore, we generated a yeast two-hybrid protein-protein interaction (PPI) network between H5N1 and human proteins. By systematically comparing the influenza-host PPI networks of three strains of IAVs, we show that different subtypes of IAVs target different human proteins and pathways, which may have led to different pathogenic phenotypes.

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