Classical Swine Fever Virus Replication Research Articles

Fatty Acid Synthase Is Involved in Classical Swine Fever Virus Replication by Interaction with NS4B.

Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, relies on host machinery to complete its life cycle. Previous studies have shown a close connection between virus infection and fatty acid biosynthesis, mainly regulated by fatty acid synthase (FASN). However, the molecular action of how FASN participates in CSFV replication remains to be elucidated. In this study, two chemical inhibitors of the fatty acid synthesis pathway [5-(tetradecyloxy)-2-furoic acid (TOFA) and tetrahydro-4-methylene-2R-octyl-5-oxo-3S-furancarboxylic acid (C75)] significantly impaired the late stage of viral propagation, suggesting CSFV replication required fatty acid synthesis. We next found that CSFV infection stimulated the expression of FASN, whereas knockdown of FASN inhibited CSFV replication. Furthermore, confocal microscopy showed that FASN participated in the formation of replication complex (RC), which was associated with the endoplasmic reticulum (ER). Interestingly, CSFV NS4B interacted with FASN and promoted overexpression of FASN, which is regulated by functional Rab18. Moreover, we found that FASN regulated the formation of lipid droplets (LDs) upon CSFV infection, promoting virus proliferation. Taken together, our work provides mechanistic insight into the role of FASN in the viral life of CSFV, and it highlights the potential antiviral target for the development of therapeutics against pestiviruses. IMPORTANCE Classical swine fever, caused by classical swine fever virus (CSFV), is one of the notifiable diseases by the World Organization for Animal Health (OIE) and causes significant financial losses to the pig industry globally. CSFV, like other (+)-strand RNA viruses, requires lipid and sterol biosynthesis for efficient replication. However, the role of lipid metabolism in CSFV replication remains unknown. Here, we found that fatty acid synthase (FASN) was involved in viral propagation. Moreover, FASN is recruited to CSFV replication sites in the endoplasmic reticulum (ER) and interacts with NS4B to regulate CSFV replication that requires Rab18. Furthermore, we speculated that lipid droplet (LD) biosynthesis, indirectly regulated by FASN, ultimately promotes CSFV replication. Our results highlight a critical role for de novo fatty acid synthesis in CSFV infection, which might help control this devastating virus.

Curcumin inhibits classical swine fever virus replication by interfering with lipid metabolism

Although previous reports have shown that Curcumin inhibits many viruses, including some important members of different genera of Flaviviridae family (Japanese encephalitis virus, dengue virus and hepatitis C virus), the antiviral activity of curcumin against Classical swine fever virus (CSFV), which belongs to Pestivirus genus, is still unclear. In this study, we found that curcumin inhibited CSFV replication in a dose-dependent manner, but had no effect on virus adsorption and entry. Furthermore, the results showed that curcumin inhibited the expression of FASN, one of the key enzymes of fatty acid synthesis pathway, thereby, causing the reduction of the production of LDs upon infection. To this end, we detected transcription factor 6 (ATF6), the key factor of regulating lipid metabolism along with other related molecules (CHOP and GPR78) and found that curcumin significantly impaired the gene synthesis of ATF6, while CSFV infection promoted ATF6 expression. Therefore, it is confirmed that curcumin inhibited CSFV replication by interfere lipid metabolism. In addition, our subsequent studies found that curcumin played an antiviral role by promoting the innate immune independent of NF-κB signaling pathway. Taken together, our finding highlights that curcumin is a potential candidate drug against CSFV for controlling CSF.

Early and Solid Protection Afforded by the Thiverval Vaccine Provides Novel Vaccination Alternatives Against Classical Swine Fever Virus.

Classical swine fever virus (CSFV) remains a challenge for the porcine industry. Inefficient vaccination programs in some endemic areas may have contributed to the emergence of low and moderate virulence CSFV variants. This work aimed to expand and update the information about the safety and efficacy of the CSFV Thiverval-strain vaccine. Two groups of pigs were vaccinated, and a contact and control groups were also included. Animals were challenged with a highly virulent CSFV strain at 21- or 5-days post vaccination (dpv). The vaccine induced rapid and strong IFN-α response, mainly in the 5-day immunized group, and no vaccine virus transmission was detected. Vaccinated pigs showed humoral response against CSFV E2 and Erns glycoproteins, with neutralising activity, starting at 14 days post vaccination (dpv). Strong clinical protection was afforded in all the vaccinated pigs as early as 5 dpv. The vaccine controlled viral replication after challenge, showing efficient virological protection in the 21-day immunized pigs despite being housed with animals excreting high CSFV titres. These results demonstrate the high efficacy of the Thiverval strain against CSFV replication. Its early protection capacity makes it a useful alternative for emergency vaccination and a consistent tool for CSFV control worldwide.

A Novel E2 Glycoprotein Subunit Marker Vaccine Produced in Plant Is Able to Prevent Classical Swine Fever Virus Vertical Transmission after Double Vaccination.

The efficacy of a novel subunit vaccine candidate, based in the CSFV E2 glycoprotein produced in plants to prevent classical swine fever virus (CSFV) vertical transmission, was evaluated. A Nicotiana benthamiana tissue culture system was used to obtain a stable production of the E2-glycoprotein fused to the porcine Fc region of IgG. Ten pregnant sows were divided into three groups: Groups 1 and 2 (four sows each) were vaccinated with either 100 μg/dose or 300 μg/dose of the subunit vaccine at 64 days of pregnancy. Group 3 (two sows) was injected with PBS. Groups 1 and 2 were boosted with the same vaccine dose. At 10 days post second vaccination, the sows in Groups 2 and 3 were challenged with a highly virulent CSFV strain. The vaccinated sows remained clinically healthy and seroconverted rapidly, showing efficient neutralizing antibodies. The fetuses from vaccinated sows did not show gross lesions, and all analyzed tissue samples tested negative for CSFV replication. However, fetuses of non-vaccinated sows had high CSFV replication in tested tissue samples. The results suggested that in vaccinated sows, the plant produced E2 marker vaccine induced the protective immunogenicity at challenge, leading to protection from vertical transmission to fetuses.

Anti-Classical Swine Fever Virus Strategies.

Classical swine fever (CSF), caused by CSF virus (CSFV), is a highly contagious swine disease with high morbidity and mortality, which has caused significant economic losses to the pig industry worldwide. Biosecurity measures and vaccination are the main methods for prevention and control of CSF since no specific drug is available for the effective treatment of CSF. Although a series of biosecurity and vaccination strategies have been developed to curb the outbreak events, it is still difficult to eliminate CSF in CSF-endemic and re-emerging areas. Thus, in addition to implementing enhanced biosecurity measures and exploring more effective CSF vaccines, other strategies are also needed for effectively controlling CSF. Currently, more and more research about anti-CSFV strategies was carried out by scientists, because of the great prospects and value of anti-CSFV strategies in the prevention and control of CSF. Additionally, studies on anti-CSFV strategies could be used as a reference for other viruses in the Flaviviridae family, such as hepatitis C virus, dengue virus, and Zika virus. In this review, we aim to summarize the research on anti-CSFV strategies. In detail, host proteins affecting CSFV replication, drug candidates with anti-CSFV effects, and RNA interference (RNAi) targeting CSFV viral genes were mentioned and the possible mechanisms related to anti-CSFV effects were also summarized.

ARFGAP1 binds to classical swine fever virus NS5A protein and enhances CSFV replication in PK-15 cells

Classical swine fever virus (CSFV), an enveloped virus belonging to the genus Pestivirus of the Flaviviridae family, utilizes cell host factors for its own replication. ARFGAP1, GTPase activating protein of ADP-ribosylation factor 1, regulates COP I vesicle formation and function in cells and is involved in the life cycle of several viruses. However, the effect of ARFGAP1 on the infection of CSFV has not been illustrated. Here we showed that inhibition of ARFGAP1 either by QS11 or by lentivirus-mediated silencing repressed CSFV replication. While, subsequent experiments revealed that CSFV production were increased in cells with sufficient ARFGAP1 expression. However, ARFGAP1 was not involved in CSFV binding, entry, access to cell vesicles, and RNA replication during the early stages of infection. Then, we showed that ARFGAP1 interacted with the viral protein of NS5A, measured by immunoprecipitation, GST-pulldown, and confocal microscopy assays. Furthermore, we revealed that ARFGAP1 could alleviated CSFV NS5A-induced endoplasmic reticulum stress (ERS). Altogether, these results demonstrate that ARFGAP1, a NS5A binding protein, is involved in CSFV replication.

Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus.

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as a cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture-adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig-expressing cells and areas of nonexpressing cells, and one single cell line revealed not to express any CD46pig The CD46pig-deficient porcine lymphoma cell line, known to facilitate CSFV replication, was the only porcine cell line nonpermissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig-independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized.IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and is highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in the absence of CD46pig Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

ABSTRACT Endoplasmic reticulum stress (ERS)-mediated autophagy is indispensable for modulation of replication and pathogenesis of numerous mammalian viruses. We have previously shown that classical swine fever virus (CSFV) infection induces ERS-mediated autophagy for maintaining viral replication both in vivo and in vitro, however, the underlying mechanism remains unclarified. Here we found that CSFV infection activates the PERK pathway-dependent complete autophagy to promote viral replication in cultured PK-15 and 3D4/2 cells. Likewise, our results also suggested the essential roles of the IRE1/GRP78-mediated complete autophagy in CSFV replication in vitro. Furthermore, we suggested that CSFV infection induces activation of the PERK and IRE1 pathway for potential immunoregulation via promoting transcription of proinflammatory cytokine (IFN-γ and TNF-α) genes in the CSFV-infected cells. Finally, pharmacological treatment of PERK- or IRE1-pathway regulators, and the corresponding SiRNAs interventions did not affect the viabilities of the cells, excluding the potential interference elicited by altered cell viabilities. Taken together, our results suggest that CSFV infection induces complete autophagy through activation of the PERK and IRE1 pathway to facilitate viral replication in cultured cells, and modulation of proinflammatory cytokines may be a potential mechanism involved in this event. Our findings will open new horizons for molecular mechanisms of sustainable replication and pathogenesis of CSFV, and lay a theoretical foundation for the development of ERS-autophagy-targeting therapeutic strategies for clinical control of CSF.

The Network of Interactions Between Classical Swine Fever Virus Nonstructural Protein p7 and Host Proteins.

Classical swine fever (CSF) is a highly contagious viral disease causing severe economic losses to the swine industry. As viroporins of viruses modulate the cellular ion balance and then take over the cellular machinery, blocking the activity of viroporin or developing viroporin-defective attenuated vaccines offers new approaches to treat or prevent viral infection. Non-structural protein p7 of CSF virus (CSFV) is a viroporin, which was highly involved in CSFV virulence. Deciphering the interaction between p7 and host proteins will aid our understanding of the mechanism of p7-cellular protein interaction affecting CSFV replication. In the present study, seven host cellular proteins including microtubule-associated protein RP/EB family member 1 (MAPRE1), voltage-dependent anion channel 1 (VDAC1), proteasome maturation protein (POMP), protein inhibitor of activated STAT 1 (PIAS1), gametogenetin binding protein 2 (GGNBP2), COP9 signalosome subunit 2 (COPS2), and contactin 1 (CNTN1) were identified as the potential interactive cellular proteins of CSFV p7 by using yeast two-hybrid (Y2H) screening. Plus, the interaction of CSFV p7 with MAPRE1 and VDAC1 was further evaluated by co-immunoprecipitation and GST-pulldown assay. Besides, the p7-cellular protein interaction network was constructed based on these seven host cellular proteins and the STRING database. Enrichment analysis of GO and KEGG indicated that many host proteins in the p7-cellular protein interaction network were mainly related to the ubiquitin-proteasome system, cGMP-PKG signaling pathway, calcium signaling pathway, and JAK-STAT pathway. Overall, this study identified potential interactive cellular proteins of CSFV p7, constructed the p7-cellular protein interaction network, and predicted the potential pathways involved in the interaction between CSFV p7 and host cells.

LDHB inhibition induces mitophagy and facilitates the progression of CSFV infection

ABSTRACT Cellular metabolism caters to the energy and metabolite needs of cells. Although the role of the terminal metabolic enzyme LDHB (lactate dehydrogenase B) in the glycolysis pathway has been widely studied in cancer cells, its role in viral infection is relatively unknown. In this study, we found that CSFV (classical swine fever virus) infection reduces pyruvate levels while promotes lactate release in pigs and in PK-15 cells. Moreover, using a yeast two-hybrid screening system, we identified LDHB as a novel interacting partner of CSFV non-structural protein NS3. These results were confirmed via co-immunoprecipitation, glutathione S-transferase and confocal assays. Furthermore, knockdown of LDHB via interfering RNA induced mitochondrial fission and mitophagy, as detected reduced mitochondrial mass. Upon inhibition of LDHB, expression of the mitophagy proteins TOMM20 and VDAC1 decreased and the ubiquitination of MFN2, a mitochondrial fusion mediator, was promoted. In addition, a sensitive dual fluorescence reporter (mito-mRFP-EGFP) was utilized to analyze the delivery of autophagosomes to lysosomes in LDHB inhibition cells. Furthermore, LDHB inhibition promoted NFKB signaling, which was regulated by mitophagy; meanwhile, infection with CSFV negated these NFKB anti-viral responses. Inhibition of LDHB also inhibited apoptosis, providing an environment conducive to persistent viral infection. Finally, we demonstrated that LDHB inhibition promoted CSFV growth via mitophagy, whereas its overexpression decreased CSFV replication. Our data revealed a novel mechanism through which LDHB, a metabolic enzyme, mediates CSFV infection, and provides new avenues for the development of anti-viral strategies.Abbreviations: 3-MA:3-methyladenine; CCCP:carbonyl cyanide 3-chlorophenylhydrazone; CCK-8:cell counting kit-8; CSFV:classical swine fever virus; DAPI:4′,6-diamidino-2-phenylindole; DMSO:dimethyl sulfoxide; EGFP:enhanced green fluorescent protein; FBS:fetal bovine serum; FITC:fluorescein isothiocyanate; GST:glutathione-S-transferase; HCV:hepatitis C virus; IFN:interferon; LDH:lactate dehydrogenase; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MFN2:mitofusin 2; MOI:multiplicity of infection; NFKB:nuclear factor kappa B subunit 1; NFKBIA:nuclear factor inhibitor alpha; NS3:nonstructural protein 3; NKIRAS2:NFKB inhibitor interacting Ras like 2; PRKN:parkin E3 ubiquitin protein ligase; PBS:phosphate-buffered saline; qRT-PCR:real-time quantitative reverse transcriptase polymerase chain reaction; RELA:RELA proto-oncogene, NF-kB subunit; shRNA: short hairpin RNA; siRNA: small interfering RNA; TCID50:50% tissue culture infectious doses; TEM:transmission electron microscopy; TNF:tumor necrosis factor; TOMM20:translocase of outer mitochondrial membrane 20; VDAC1:voltage dependent anion channel 1.

Antiviral Role of Serine Incorporator 5 (SERINC5) Proteins in Classical Swine Fever Virus Infection.

Serine incorporator 5 (SERINC5), a multipass transmembrane protein, protects cells from viral infections. The mechanism by which SERINC5 protects against classical swine fever virus (CSFV) infection is unknown. In this study, overexpression of SERINC5 in PK-15 and 3D4/2 cells significantly inhibited the growth of CSFV, whereas SERINC5 silencing enhanced CSFV growth. Additionally, CSFV infection reduced SERINC5 production in cells and tissues. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify and analyze protein and peptide molecules that potentially interact with SERINC5. A total of 33 cellular protein candidates were identified. Next, SERINC5 was shown to interact with melanoma differentiation-associated protein 5 (MDA5) by yeast two-hybrid, protein co-localization and co-immunoprecipitation assays. Furthermore, SERINC5 enhanced MDA5-mediated type I interferon (IFN) signaling in a dose-dependent manner. Our results suggest that the anti-CSFV effect of SERINC5 is dependent on the activation of the type I IFN, which may function along with MDA5. The inhibitory effect of SERINC5 on CSFV was disappeared when the endogenous expression of MDA5 was silenced using siRNA, suggesting that SERINC5 exerts an anti-CSFV effect in an MDA5-dependent manner. Our study demonstrated a novel link between SERINC5 and MDA5 in the inhibition of CSFV replication via the type I IFN signaling pathway.

Isolation and propagation of classical swine fever virus in porcine Wharton’s Jelly mesenchymal stem cells

Classical Swine Fever (CSF) is an extremely infectious and deadly disease of pigs and wild boars caused by the CSF virus (CSFV) which is a member of the Pestivirus genus and the family Flaviviridae. This study was designed to detect the permissibility and replication of CSFV in mesenchymal stem cells (MSCs) monolayer derived from Porcine Wharton’s jelly. Porcine Wharton’s jelly MSCs (pWJ-MSCs) were ex vivo expanded and propagated for more than 81 generations and third passage pWJ-MSCs were characterized as per standard criteria i.e., growth characteristics, trilineage differentiation potential and molecular characterization for pluripotency and stem cell surface markers. Porcine WJ tissue samples found negative for CSFV by RT-PCR test were processed further for the isolation of pWJ-MSCs and CSFV was propagated over the characterized pWJ-MSCs monolayer. No cytopathic effect was observed, which was consistent with non-cytopathic nature of CSFV. The replication of CSFV in pWJ-MSCs was affirmed by RT-PCR and demonstration of viral antigen in the cytoplasm of virus infected cells by immuno-staining technique. In total, three different CSFV isolates were propagated in pWJ-MSCs. Primary pWJ-MSCs permitted CSFV replication to good titer. To the best of our information, this is the first ever report of isolation of CSFV in pWJ-MSCs.

MG132 Attenuates the Replication of Classical Swine Fever Virus in vitro.

The 26S proteasome, in charge of intracellular protein degradation, plays significant roles in the modulation of various cellular activities as well as in the interplay between virus and host. However, studies about the relationship between 26S proteasome and classical swine fever virus (CSFV) is limited up to now. MG132 is a proteasome inhibitor and has been extensively used in studies about replication of many viruses. Herein, we investigated the role of MG132 in CSFV replication and results showed that MG132 significantly decreased virus titers and viral RNA copies in CSFV-infected PK-15 cells. Further studies demonstrated that MG132 upregulated the expression of several interferon-stimulated genes (ISGs), in CSFV-infected cells. Since the activation of ISGs is controlled by the JAK-STAT signal pathway, we next examined the effect of MG132 on the expression and localization of key molecular STAT1 in the infected cells using Western blot and confocal laser scanning microscopy, respectively. Results showed that CSFV infection and viral NS4A protein decreased the protein level of STAT1, and MG132 promoted the accumulation of STAT1 in the nucleus of cells adjacent to the CSFV-infected cells. Besides, MG132 did not affect the expressions of IFN-α, STAT1, Mx1, OAS1, and PKR genes in cells without CSFV. In conclusion, we identify that MG132 significantly inhibits CSFV replication in vitro, in which the activation of the JAK-STAT pathway and the subsequent upregulation of expressions of ISGs might play significant roles, providing a potential preventive method for CSF.

Rab1b-GBF1-ARFs mediated intracellular trafficking is required for classical swine fever virus replication in swine umbilical vein endothelial cells

Classical swine fever virus (CSFV), a plus-sense RNA virus, utilizes host intracellular membrane organelles for its replication. Our previous studies have shown that disruption of the intracellular membrane-trafficking events can inhibit CSFV replication. However, the underlying mechanism of this process in CSFV infection has not been elucidated. To determine the role of Golgi-associated anterograde and retrograde trafficking in CSFV replication, we revealed the effect of vesicular transport between Golgi and ER inhibitors Brefeldin A (BFA) and 2,2-methyl-N-(2,4,6,-trimethoxyphenyl) dodecanamide (CI-976), the GBF1 inhibitor golgicide A (GCA) on virus production. Our results showed that disruption of vesicular trafficking by BFA, CI-976, and GCA significantly inhibited CSFV infection. Subsequent experiments revealed that knockdown of Rab1b by lentiviruses and negative-mutant Rab1b-N121I transfection inhibited CSFV infection. Furthermore, we showed that the Rab1b downstream vesicular component effectors GBF1, and class I and class II ADP-ribosylation factors (ARFs) were also involved in virus replication. In addition, confocal microscopy assay showed that CSFV infection disrupted the Golgi apparatus resulting in extended Golgi distribution around the nucleus. We also showed that cell secretory pathway, measured using Gaussia luciferase flash assay, was blocked in CSFV infected cells. Taken together, these findings demonstrate that CSFV utilizes Rab1b-GBF1-ARFs mediated trafficking to promote its own replication. These findings also provide new insights into the intracellular trafficking pathways utilized for CSFV life cycle.

Induction of autophagy and suppression of type I IFN secretion by CSFV

ABSTRACT Macroautophagy/autophagy plays an essential role in cellular responses to pathogens. However, the precise mechanisms and signaling pathways that modulate cellular autophagy in classical swine fever virus (CSFV)-infected host cells have not been confirmed. In this study, we showed that CSFV infection inhibits the phosphorylation of MTOR (mechanistic target of rapamycin kinase), subsequently leading to autophagy initiation. We also show that MAPK/ERK (mitogen-activated protein kinase) signaling is involved in CSFV-induced autophagy. The CSFV-induced inhibition of AKT/PKB (AKT serine/threonine kinase)-MTOR was observed to be partially responsible for the MTOR inactivation and subsequent autophagy initiation. Moreover, the CAMKK2/CaMKKβ (calcium/calmodulin dependent protein kinase kinase 2)-PRKAA/AMPK (protein kinase AMP-activated catalytic subunit alpha) axis was found to be involved in CSFV-induced autophagy. Meanwhile, CSFV non-structural protein NS5A induced autophagy via the CAMKK2-PRKAA-MTOR signaling pathway but not the AKT-MTOR or MAPK1/ERK2-MAPK3/ERK1-MTOR pathway. Although the AKT-MTOR pathway also plays an important role in the induction of autophagy by CSFV. We also found the interaction between HSP90AB1/HSPCB and NS5A by tandem affinity purification/liquid chromatography-mass spectrometry (LC-MS) and immunoprecipitation. Furthermore, the CSFV-induced [Ca2+]cyto increase potently induced autophagy through CAMKK2 and PRKAA. Moreover, we isolated and identified the BECN1/Beclin 1 protein complexes by tandem affinity purification/LC-MS and immunoprecipitation, the interaction between BECN1 and MAVS was confirmed by immunoprecipitation, laser scanning confocal microscope technology, and GST affinity-isolation experiments. Furthermore, CSFV-mediated autophagy suppressing type I IFN production is related to the interaction between MAVS and BECN1. Finally, the modulation of autophagy induction pathways by different autophagy regulatory factors significantly affected the replication of CSFV. Abbreviations: AKT: AKT serine/threonine kinase; AMPK: Adenosine monophosphate-activated protein kinase; CAMKK2: Calcium/calmodulin dependent protein kinase kinase 2; CSFV: Classical swine fever virus; HRP: Horseradish peroxidase; HSP90AB1: Heat shock protein 90 alpha family class B member 1; IFN: Interferon; ISGs: IFN-stimulated genes; LC-MS: Liquid chromatography-mass spectrometry; MAP1LC3/LC3: Microtubule associated protein 1 light chain 3; MAPK: Mitogen-activated protein kinase; MAVS: Mitochondrial antiviral signaling protein; MOI: Multiplicity of infection; MTOR: Mechanistic target of rapamycin kinase; PBS: Phosphate-buffered saline; PRKAA: Protein kinase AMP-activated catalytic subunit alpha; shRNA: short hairpin RNA.

MiR-140 inhibits classical swine fever virus replication by targeting Rab25 in swine umbilical vein endothelial cells

ABSTRACT Classical swine fever virus (CSFV) is one of the most important viral pathogens leading worldwide threats to pig industry. MicroRNAs (miRNAs) play important roles in regulating virus replication, but whether miRNAs affect CSFV infection is still poorly understood. In previous study, we identified four miRNAs that were down-regulated by CSFV in swine umbilical vein endothelial cells (SUVEC). In this study, miR-140, one of the most potently down-regulated genes was investigated. We found that the miRNA expression was significantly inhibited by CSFV infection. Subsequent studies revealed that miR-140 mimics significantly inhibited CSFV replication, while the inhibition of endogenous miR-140 enhanced CSFV replication. By using bioinformatics prediction, luciferase reporter system, real-time fluorescence quantitative PCR (RT-qPCR) and Western blot assays, we further demonstrated that miR-140 bind to the 3ʹ UTR of Rab25 mRNA to regulate its expression. We also analyzed the expression pattern of Rab25 in SUVECs after CSFV infection. The results showed that CSFV infection induced Rab25 expression. Finally, Rab25 was found to promote CSFV replication. In conclusion, this study demonstrated that CSFV inhibits miR-140 expression and miR-140 inhibits replication by binding to host factor Rab25.

The R614E mutation of mouse Mx1 protein contributes to the novel antiviral activity against classical swine fever virus

Mx proteins are interferon-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses. We previously demonstrated that porcine Mx1 protein (poMx1) inhibited the replication of classical swine fever virus (CSFV), an economically important Pestivirus, and that mouse Mx1 did so as well. It is unknown why the nucleus-localizing mouse Mx1 inhibits CSFV replication which occurs in the cytoplasm. To the end, we assessed the anti-CSFV actions of wild type mouse Mx1 and seven previously reported mutants (K49A, G83R, A222V, A516V, G540E, R614E and ΔL4) and identified the molecular mechanism of R614E action against CSFV replication. A series of experiments revealed that mmMx1 (R614E) mutant reposted to the cytoplasm and interacted with the CSFV nucleocapsid protein (Core), thereby inhibiting viral replication. These findings broaden our understanding of the function of Mx protein family members against CSFV and suggest that the relative conservation of Mx1 among species is the basis of broad-spectrum antiviral properties.

Antiviral activity of ISG15 against classical swine fever virus replication in porcine alveolar macrophages via inhibition of autophagy by ISGylating BECN1

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs) for defense against numerous viral infections, including classical swine fever virus (CSFV). However, the mechanisms underlying the effect of ISGs on CSFV infection are rarely reported. In this study, we demonstrate that IFN-α treatment induces upregulation of ISG15 and thus attenuates CSFV replication. To determine whether ISG15 is critical for controlling CSFV replication, we established porcine alveolar macrophages (PAMs) with stable overexpression or knockdown of ISG15. Overexpression of Flag-ISG15 significantly prevented CSFV replication, whereas loss of ISG15 led to abnormal proliferation of CSFV. Furthermore, upregulated ISG15 promoted beclin-1 (BECN1) ISGylation and dysfunction and subsequently inhibited autophagy, which is indispensable for CSFV replication. In addition, HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), which functions to catalyze conjugation of ISG15 protein, was confirmed to interact with BECN1. Collectively, these results indicate that IFN-α restricts CSFV replication through ISG15-mediated BECN1 ISGylation and autophagy inhibition, providing insight into the mechanism of CSFV replication control by type I IFN. This mechanism may not be the only antiviral mechanism of ISG15; nonetheless, this study may contribute to the development of CSFV treatment and prevention strategies.

Dual NDP52 Function in Persistent CSFV Infection.

Viruses have evolved many mechanisms to escape host antiviral responses. Previously, we found that classical swine fever virus (CSFV) infection induces autophagy using the autophagosome as a self-replication site, thereby evading the host immune response and promoting long-term infection. However, the underlying mechanisms used by CSFV to enter autophagosomes and the mechanism by which autophagy promotes viral replication remain unclear. We found that CSFV infection inhibited autophagy receptor nuclear dot protein 52 kDa (NDP52) expression, ubiquitination, and SUMO2-4 modification. Further analyses revealed that CSFV mediated ubiquitination and SUMOylation of NDP52 via Pten-induced kinase 1 (PINK1)-Parkin. Moreover, NDP52 inhibition also inhibited CSFV replication and the induction of mitophagy marker proteins expression. Inhibition of NDP52 reduced CD63 expression and binding to CSFV E2 protein, which has an essential role in persistent CSFV infection. As NDP52 has a close relationship with the NF-κB innate immunity pathway and plays an important role in the antiviral response, we investigated whether NDP52 inhibited CSFV replication through the release of immune factors and antivirus signals. Our results showed that inhibiting NDP52 boosted interferon and TNF release and promoted NF-κB pathway activation. In summary, we found that NDP52 inhibition not only reduces CSFV binding and entry into autophagic vesicles, but also inhibits CSFV replication by active NF-κB antiviral immune pathways. Our data reveal a novel mechanism by which NDP52, an autophagy receptor, mediates CSFV infection, and provide new avenues for the development of antiviral strategies.

CSFV protein NS5A activates the unfolded protein response to promote viral replication

Classical swine fever is a world organization for animal health listed disease and is caused by classical swine fever virus (CSFV). CSFV can induced unfolded protein response (UPR) and whether NS5A protein plays a role in this process remains unknown. Here, we demonstrate that CSFV induced all the three signal pathways ATF6, IRE1 and PERK of UPR. Furthermore, this phenomenon may be mediated by the NS5A protein since expression of NS5A alone can achieve the same effect. In the current study, we show that NS5A can interact with GRP78 as measured by using the CO-IP and GST pulldown assays. This interaction plays a positive role in the promotion of CSFV replication. Overexpression or knockdown of GRP78 mediated by lentivirus can enhance or decrease viral replication, respectively. Our findings provide the evidence that CSFV infection can activate the cellular UPRs, in which NS5A and GRP78 play key roles in the process.