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

Abstract

The 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 glutathione S-transferase (GST) pulldown, in situ proximity 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. C lassical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious, often fatal porcine disease with significant economic losses. CSFV belongs to the Pestivirus genus within the Flaviviridae family (1). It is an enveloped virus with a single-stranded, positive-sense RNA genome of approximately 12.3 kb in length. The RNA genome contains a single large open reading frame (ORF). This ORF is translated into a polyprotein, which is further processed into 12 mature proteins (N pro -C-E rns -E1-E2-p7-NS2-3-NS4A-NS4B-NS5A-NS5B) by viral and cellular proteases (2). The C, E rns , E1, and E2 proteins represent the structural components of the virion. The E1 and E2 glycoproteins are anchored to the envelope by their carboxyl termini, while E rns is loosely associated with the envelope. E rns presents as homodimers linked by disulfide bridges in virus-infected cells and in virions (3, 4). The C terminus of E2 functions as a membrane-spanning domain anchoring the E2-E1 or E2-E2 dimer to the viral envelope (5). E2 is also involved in virus attachment to and entry into the target cell (6). Furthermore, as a virulence determinant in pigs (7), the E2 protein can efficiently induce protective immune responses (8-13). Additionally, we recently showed that host ␤-actin interacts with E2 and modulates the early life cycle of CSFV (14).Thioredoxins (Trxs) are a class of ubiquitously expressed redox proteins that contain a conserved consensus amino acid sequence (Cys-Gly-Pro-Cys) in the catalytic center. There are two distinct forms of Trxs (Trx1 and Trx2), which act as antioxidants facilitating the reduction of other proteins by cysteine thiol-disulfide exchange. Trx2 consists of 157 amino acid (aa) residues, and its thioredoxin domain is located in the C terminus of the protein.Trxs are involved in diverse biological processes, such as cell growth, proliferation, apoptosis, and gene regulation (15-18). Additionally, they interact with several transcription factors and

Mitogen-Activated Protein Kinase Kinase 2, a Novel E2-Interacting Protein, Promotes the Growth of Classical Swine Fever Virus via Attenuation of the JAK-STAT Signaling Pathway

“…12007C; Sigma-Aldrich) and maintained at 37°C in 5% CO 2 . The CSFV Shimen strain was propagated in PK-15 cells as described previously (13) and titrated using the Reed-Muench formula (29). …”

“…The BD-E2 construct was used as bait to hybridize with a porcine primary macrophage cDNA library (13). Transformants were screened on the plates containing synthetically defined medium lacking Leu, Trp, His, and Ade (SD/−4) for 4 to 6 days.…”

“…Positive colonies were further identified on SD/−4 medium containing 5-bromo-4-chloro-3-indolyl-α- d -galactopyranoside (X-α-Gal) and aureobasidin A (Aba) (SD/−4/X/Aba). The positive colonies were cultured in the SD/−4 medium and verified by sequencing as described previously (13). To validate the interaction between E2 and MEK2, the Y2HGold yeast strain was cotransformed with the plasmids BD-E2 and pGADT7-MEK2 (AD-MEK2) using yeast transformation system 2 (catalog no.…”

“…In addition, E2 is a major determinant for virus virulence and host tropism (10). In fact, several E2-interacting host cellular proteins, including β-actin (11), annexin 2 (12), and thioredoxin 2 (13), have been identified to play important roles in the virus life cycle.…”

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

“…E rns has been demonstrated to interact with the Laminin receptor [15]. In addition, E2 has been shown to interact with several host proteins including cellular actin [16], thioredoxin [17], annexin 2 [18], mitogen-activated protein kinase 2 [19], protein phosphatase 1 catalytic subunit beta [20] and dynactin 6 [21]. These host-virus protein-protein interactions have been shown to play a role in regulating the virus replication cycle and, in some cases, these protein interactions are involved in virus virulence [11][12][13]21].…”