Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious, economically important viral disease in many countries. The E rns and E2 envelope glycoproteins are responsible for the binding to and entry into the host cell by CSFV. To date, only one cellular receptor, heparan sulfate (HS), has been identified as being involved in CSFV attachment. HS is also present on the surface of various cells that are nonpermissive to CSFV. Hence, there must be another receptor(s) that has been unidentified to date. In this study, we used a set of small interfering RNAs (siRNAs) against a number of porcine cell membrane protein genes to screen cellular proteins involved in CSFV infection. This approach resulted in the identification of several proteins, and of these, the laminin receptor (LamR) has been demonstrated to be a cellular receptor for several viruses. Confocal analysis showed that LamR is colocalized with CSFV virions on the membrane, and a coimmunoprecipitation assay indicated that LamR interacts with the CSFV E rns protein. In inhibition assays, anti-LamR antibodies, soluble laminin, or LamR protein significantly inhibited CSFV infection in a dose-dependent manner. Transduction of PK-15 cells with a recombinant lentivirus expressing LamR yielded higher viral titers. Moreover, an attachment assay demonstrated that LamR functions during virus attachment. We also demonstrate that LamR acts as an alternative attachment receptor, especially in SK6 cells. These results indicate that LamR is a cellular attachment receptor for CSFV. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only heparan sulfate (HS) has been identified to be an attachment receptor for CSFV. Here, using RNA interference screening with small interfering RNAs (siRNAs) against a number of porcine membrane protein genes, we identified the laminin receptor (LamR) to be another attachment receptor. We demonstrate the involvement of LamR together with HS in virus attachment, and we elucidate the relationship between LamR and HS. LamR also serves as an attachment receptor for many viral pathogens, including dengue virus, a fatal human flavivirus. The study will help to enhance our understanding of the life cycle of flaviviruses and the development of antiviral strategies for flaviviruses. C lassical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and often fatal viral disease in pigs. The disease leads to significant economic losses in many countries. CSFV is a member of the Pestivirus genus within the Flaviviridae family (1). The virus possesses a singlestranded, positive-sense RNA genome of approximately 12.3 kb (2, 3). Its genome contains a single, large open reading frame that encodes a precursor polyprotein of 3,898 amino acids (aa). The polyprotein is co-and posttranslationally processed by viral an...

The mechanisms on metabolic regulation of immune responses are still elusive. We show here that viral infection induces immediate-early NF-κB activation independent of viral nucleic acid-triggered signaling, which triggers a rapid transcriptional induction of bile acid (BA) transporter and rate-limiting biosynthesis enzymes as well as accumulation of intracellular BAs in divergent cell types. The accumulated intracellular BAs activate SRC kinase via the TGR5-GRK-β-arrestin axis, which mediates tyrosine phosphorylation of multiple antiviral signaling components including RIG-I, VISA/MAVS, MITA/STING, TBK1 and IRF3. The tyrosine phosphorylation of these components by SRC conditions for efficient innate antiviral immune response. Consistently, TGR5 deficiency impairs innate antiviral immunity, whereas BAs exhibit potent antiviral activity in wild-type but not TGR5-deficient cells and mice. Our findings reveal an intrinsic and universal role of intracellular BA metabolism in innate antiviral immunity.

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

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The mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK1/2/ERK1/2) cascade is involved in the replication of several members of the Flaviviridae family, including hepatitis C virus and dengue virus. The effects of the cascade on the replication of classical swine fever virus (CSFV), a fatal pestivirus of pigs, remain unknown. In this study, MEK2 was identified as a novel binding partner of the E2 protein of CSFV using yeast two-hybrid screening. The E2-MEK2 interaction was confirmed by glutathione S-transferase pulldown, coimmunoprecipitation, and laser confocal microscopy assays. The C termini of E2 (amino acids [aa] 890 to 1053) and MEK2 (aa 266 to 400) were mapped to be crucial for the interaction. Overexpression of MEK2 significantly promoted the replication of CSFV, whereas knockdown of MEK2 by lentivirus-mediated small hairpin RNAs dramatically inhibited CSFV replication. In addition, CSFV infection induced a biphasic activation of ERK1/2, the downstream signaling molecules of MEK2. Furthermore, the replication of CSFV was markedly inhibited in PK-15 cells treated with U0126, a specific inhibitor for MEK1/2/ERK1/2, whereas MEK2 did not affect CSFV replication after blocking the interferon-induced Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway by ruxolitinib, a JAK-STAT-specific inhibitor. Taken together, our results indicate that MEK2 positively regulates the replication of CSFV through inhibiting the JAK-STAT signaling pathway.IMPORTANCE Mitogen-activated protein kinase kinase 2 (MEK2) is a kinase that operates immediately upstream of extracellular regulated kinase 1/2 (ERK1/2) and links to Raf and ERK via phosphorylation. Currently, little is known about the role of MEK2 in the replication of classical swine fever virus (CSFV), a devastating porcine pestivirus. Here, we investigated the roles of MEK2 and the MEK2/ERK1/2 cascade in the growth of CSFV for the first time. We show that MEK2 positively regulates CSFV replication. Notably, we demonstrate that MEK2 promotes CSFV replication through inhibiting the interferon-induced JAK-STAT signaling pathway, a key antiviral pathway involved in innate immunity. Our work reveals a novel role of MEK2 in CSFV infection and sheds light on the molecular basis by which pestiviruses interact with the host cell.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating disease of swine worldwide. Although a mandatory vaccination with the modified live vaccine C-strain has been implemented in China for decades, CSF remains a serious threat to the swine industry. To facilitate the control and eradication of CSF in China, the E2-based marker vaccine rAdV-SFV-E2, an adenovirus-delivered, alphavirus replicon-vectored vaccine, has been developed. Accordingly, an accompanying discriminatory test that allows differentiating infected from vaccinated animals (DIVA) is required. Here, the enhanced expression of E(rns) protein of CSFV was achieved in the methyltropic yeast Pichia pastoris by codon-optimization of the E(rns) gene, and an indirect enzyme-linked immunosorbent assay (iELISA) based on the yeast-expressed E(rns) (yE(rns)) was developed and evaluated. The optimized iELISA was able to detect CSFV-specific antibodies in the serum samples from the CSFV-infected pigs as early as 6 days post-infection, and discriminate the CSFV-infected pigs from those vaccinated with rAdV-SFV-E2. The iELISA was evaluated using a panel of swine sera, and showed comparable sensitivity (94.6%) and specificity (97.1%), and the consistence rates with the virus neutralization test were 96.8% for CSFV-infected swine sera, 83.3% for C-strain-vaccinated swine sera, and 95.0% for field swine sera. In addition, the iELISA showed higher sensitivity (90.4%) compared with PrioCHECK CSFV E(rns) (59.6%). Taken together, the yE(rns)-based iELISA is specific and sensitive, representing a promising DIVA test for E2-based marker vaccines against CSF.

African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and fatal disease found in swine. However, the viral proteins and mechanisms responsible for immune evasion are poorly understood, which has severely hindered the development of vaccines. This review mainly focuses on studies involving the innate antiviral immune response of the host and summarizes the latest studies on ASFV genes involved in interferon (IFN) signaling and inflammatory responses. We analyzed the effects of candidate viral proteins on ASFV infection, replication and pathogenicity and identified potential molecular targets for novel ASFV vaccines. These efforts will contribute to the construction of novel vaccines and wonder therapeutics for ASF.

The NS5A protein of classical swine fever virus (CSFV) is involved in the RNA synthesis and viral replication. However, the NS5A-interacting cellular proteins engaged in the CSFV replication are poorly defined. Using yeast two-hybrid screen, the eukaryotic elongation factor 1A (eEF1A) was identified to be an NS5A-binding partner. The NS5A–eEF1A interaction was confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pulldown and laser confocal microscopy assays. The domain I of eEF1A was shown to be critical for the NS5A–eEF1A interaction. Overexpression of eEF1A suppressed the CSFV growth markedly, and conversely, knockdown of eEF1A enhanced the CSFV replication significantly. Furthermore, eEF1A, as well as NS5A, was found to reduce the translation efficiency of the internal ribosome entry site (IRES) of CSFV in a dose-dependent manner, as demonstrated by luciferase reporter assay. Streptavidin pulldown assay revealed that eEF1A could bind to the CSFV IRES. Collectively, our results suggest that eEF1A interacts with NS5A and negatively regulates the growth of CSFV.