Uncovering the Interaction between TRAF1 and MAVS in the RIG-I Pathway to Enhance the Upregulation of IRF1/ISG15 during Classical Swine Fever Virus Infection.

Classical swine fever (CSF) is caused by the classical swine fever virus (CSFV), which poses a threat to swine production. The activation of host innate immunity through linker proteins such as tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) is crucial for the induction of the NF-κB pathway. Recent research has revealed the involvement of mitochondrial antiviral-signaling protein (MAVS) in the interaction with TRAF2, 3, 5, and 6 to activate both the NF-κB and IRF3 pathways. This study revealed that CSFV infection led to the upregulation of TRAF1 mRNA and protein levels; moreover, TRAF1 overexpression inhibited CSFV replication, while TRAF1 knockdown promoted replication, highlighting its importance in the host response to CSFV infection. Additionally, the expression of RIG-I, MAVS, TRAF1, IRF1, and ISG15 were detected in PK-15 cells infected with CSFV, revealing that TRAF1 plays a role in regulating IRF1 and ISG15 within the RIG-I pathway. Furthermore, Co-IP, GST pull-down, and IFA analyses demonstrated that TRAF1 interacted with MAVS and co-localized in the cytoplasm during CSFV infection. Ultimately, TRAF1 acted as a novel member of the TRAF family, bound to MAVS as a linker molecule, and functioned as a mediator downstream of MAVS in the RIG-I/MAVS pathway against CSFV replication.

Classical swine fever virus non-structural protein 4A recruits dihydroorotate dehydrogenase to facilitate viral replication.

Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.

PKM2 induces mitophagy through the AMPK-mTOR pathway promoting CSFV proliferation.

Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.

CSFV restricts necroptosis to sustain infection by inducing autophagy/mitophagy-targeted degradation of RIPK3.

IMPORTANCE: CSFV infection in pigs causes persistent high fever, hemorrhagic necrotizing multi-organ inflammation, and high mortality, which seriously threatens the global swine industry. Cell death is an essential immune response of the host against pathogen invasion, and lymphopenia is the most typical clinical feature in the acute phase of CSFV infection, which affects the initial host antiviral immunity. As an "old" virus, CSFV has evolved mechanisms to evade host immune response after a long genetic evolution. Here, we show that necroptosis is a limiting host factor for CSFV infection and that CSFV-induced autophagy can subvert this host defense mechanism to promote its sustained replication. Our findings reveal a complex link between necroptosis and autophagy in the process of cell death, provide evidence supporting the important role for CSFV in counteracting host cell necrosis, and enrich our knowledge of pathogens that may subvert and evade this host defense.

Host cell factors involved in classical swine fever virus entry.

Classical swine fever virus (CSFV) is an ancient pathogen that continues to pose a threat to animal agriculture worldwide. The virus belongs to the genus Pestivirus and the family Flaviviridae. It causes a multisystemic disease that affects only pigs and is responsible for significant economic losses. CSFV infection is probably a multistep process that involves the proteins in the virus envelope and more than one receptor in the membrane of permissive cells. To date, the cellular receptors essential for CSFV entry and their detailed functions during this process remains unknown. All the viral envelope proteins Erns, E1 and E2 are involved in the entry process to some extent and the experimental approaches conducted until now have helped to unveil their contributions. This review aims to provide an overview of current knowledge on cellular molecules described to be involved in CSFV entry, including complement regulatory protein 46 (CD46), heparan sulphate (HS), Laminin receptor, Integrin ß3, Annexin II, MERKT and ADAM17. This knowledge would not only help to understand the molecular mechanisms involved in pestivirus infection, but also provide a rational basis for the development of nonvaccinal alternatives for CSFV control.

Classical swine fever virus NS5A protein activates autophagy via the PP2A-DAPK3-Beclin 1 axis.

IMPORTANCE: Autophagy is a conserved degradation process that maintains cellular homeostasis and regulates native and adaptive immunity. Viruses have evolved diverse strategies to inhibit or activate autophagy for their benefit. The paper reveals that CSFV NS5A mediates the dissociation of PP2A from Beclin 1 and the association of PP2A with DAPK3 by interaction with PPP2R1A and DAPK3, PP2A dephosphorylates DAPK3 to activate its protein kinase activity, and activated DAPK3 phosphorylates Beclin 1 to trigger autophagy, indicating that NS5A activates autophagy via the PP2A-DAPK3-Beclin 1 axis. These data highlight a novel mechanism by which CSFV activates autophagy to favor its replication, thereby contributing to the development of antiviral strategies.

Classical Swine Fever Virus Structural Glycoprotein E2 Interacts with Host Protein ACADM during the Virus Infectious Cycle.

The E2 glycoprotein is one of the four structural proteins of the classical swine fever virus (CSFV) particle. E2 has been shown to be involved in many virus functions, including adsorption to host cells, virus virulence and interaction with several host proteins. Using a yeast two-hybrid screen, we have previously shown that the CSFV E2 specifically interacts with swine host protein medium-chain-specific acyl-Coenzyme A dehydrogenase (ACADM), an enzyme that catalyzes the initial step of the mitochondrial fatty acid beta-oxidation pathway. Here, we show that interaction between ACADM and E2 also happens in swine cells infected with CSFV using two different procedures: coimmunoprecipitation and a proximity ligation assay (PLA). In addition, the amino acid residues in E2 critically mediating the interaction with ACADM, M49 and P130 were identified via a reverse yeast two-hybrid screen using an expression library composed of randomly mutated versions of E2. A recombinant CSFV, E2ΔACADMv, harboring substitutions at residues M49I and P130Q in E2, was developed via reverse genomics from the highly virulent Brescia isolate. E2ΔACADMv was shown to have the same kinetics growth in swine primary macrophages and SK6 cell cultures as the parental Brescia strain. Similarly, E2ΔACADMv demonstrated a similar level of virulence when inoculated to domestic pigs as the parental Brescia. Animals intranasally inoculated with 105 TCID50 developed a lethal form of clinical disease with virological and hematological kinetics changes undistinguishable from those produced by the parental strain. Therefore, interaction between CSFV E2 and host ACADM is not critically involved in the processes of virus replication and disease production.

The Small GTPase Rab14 Regulates the Trafficking of Ceramide from Endoplasmic Reticulum to Golgi Apparatus and Facilitates Classical Swine Fever Virus Assembly.

Classical swine fever virus (CSFV) is a highly pathogenic RNA virus belonging to the Flaviviridae family that can cause deadly classical swine fever (CSF) in pigs. However, the molecular details of virus replication in the host are still unclear. Our previous studies have reported that several Rab proteins mediate CSFV entry into host cells, but it is unknown whether CSFV hijacks other Rab proteins for effective viral infection. Here, we systematically studied the role of Rab14 protein in regulating lipid metabolism for promoting viral assembly. First, Rab14 knockdown and overexpression significantly affected CSFV replication, indicating the essential role of Rab14 in CSFV infection. Interestingly, Rab14 could significantly affect virus replication in the late stage of infection. Mechanistically, CSFV NS5A recruited Rab14 to the ER, followed by ceramide transportation to the Golgi apparatus, where sphingomyelin was synthesized. The experimental data of small molecule inhibitors, RNA interference, and replenishment assay showed that the phosphatidylinositol-3-kinase (PI3K)/AKT/AS160 signaling pathway regulated the function of Rab14 to affect the transport of ceramide. More importantly, sphingomyelin on the Golgi apparatus contributed to the assembly of viral particles. Blockage of the Rab14 regulatory pathway induced the reduction of the content of sphingomyelin on the Golgi apparatus, impairing the assembly of virus particles. Our study clarifies that Rab14 regulates lipid metabolism and promotes CSFV replication, which provides insight into a novel function of Rab14 in regulating vesicles to transport lipids to the viral assembly factory. IMPORTANCE The Rab protein family members participate in the viral replication of multiple viruses and play important roles in the virus infection cycle. Our previous research focused on Rab5/7/11, which regulated the trafficking of vesicles in the early stage of CSFV infection, especially in viral endocytosis. However, the role of other Rab proteins in CSFV replication is unclear and needs further clarification. Strikingly, we screened some Rabs and found the important role of Rab14 in CSFV infection. Virus infection mobilized Rab14 to regulate the vesicle to transport ceramide from the ER to the Golgi apparatus, further promoting the synthesis of sphingomyelin and facilitating virus assembly. The treatment of inhibitors showed that the lipid transport mediated by Rab14 was regulated by the PI3K/AKT/AS160 signaling pathway. Knockdown of Rab14 or the treatment with PI3K/AKT/AS160 inhibitors reduced the ceramide content in infected cells and hindered virus assembly. Our study is the first to explain that vesicular lipid transport regulated by Rab promotes CSFV assembly, which is conducive to the development of antiviral drugs.

Intracellular Vimentin Regulates the Formation of Classical Swine Fever Virus Replication Complex through Interaction with NS5A Protein.

Vimentin (VIM), an indispensable protein, is responsible for the formation of intermediate filament structures within cells and plays a crucial role in viral infections. However, the precise role of VIM in classical swine fever virus (CSFV) infection remains unclear. Herein, we systematically investigated the function of VIM in CSFV replication. We demonstrated that both knockdown and overexpression of VIM affected CSFV replication. Furthermore, we observed by confocal microscopy the rearrangement of cellular VIM into a cage-like structure during CSFV infection. Three-dimensional (3D) imaging indicated that the cage-like structures were localized in the endoplasmic reticulum (ER) and ringed around the double-stranded RNA (dsRNA), thereby suggesting that VIM was associated with the formation of the viral replication complex (VRC). Mechanistically, phosphorylation of VIM at serine 72 (Ser72), regulated by the RhoA/ROCK signaling pathway, induced VIM rearrangement upon CSFV infection. Confocal microscopy and coimmunoprecipitation assays revealed that VIM colocalized and interacted with CSFV NS5A. Structurally, it was determined that amino acids 96 to 407 of VIM and amino acids 251 to 416 of NS5A were the respective important domains for this interaction. Importantly, both VIM knockdown and disruption of VIM rearrangement inhibited the localization of NS5A in the ER, implying that VIM rearrangement recruited NS5A to the ER for VRC formation. Collectively, our results suggest that VIM recruits NS5A to form a stable VRC that is protected by the cage-like structure formed by VIM rearrangement, ultimately leading to enhanced virus replication. These findings highlight the critical role of VIM in the formation and stabilization of VRC, which provides alternative strategies for the development of antiviral drugs. IMPORTANCE Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly infectious disease that poses a significant threat to the global pig industry. Therefore, gaining insights into the virus and its interaction with host cells is crucial for developing effective antiviral measures and controlling the spread of CSF. Previous studies have shown that CSFV infection induces rearrangement of the endoplasmic reticulum, leading to the formation of small vesicular organelles containing nonstructural protein and double-stranded RNA of CSFV, as well as some host factors. These organelles then assemble into viral replication complexes (VRCs). In this study, we have discovered that VIM recruited CSFV NS5A to form a stable VRC that was protected by a cage-like structure formed by rearranged VIM. This enhanced viral replication. Our findings not only shed light on the molecular mechanism of CSFV replication but also offer new insights into the development of antiviral strategies for controlling CSFV.

25-hydroxycholesterol inhibits classical swine fever virus entry into porcine alveolar macrophages by depleting plasma membrane cholesterol.

Classical swine fever virus (CSFV) is an enveloped positive-sense RNA virus belonging to the Flaviviridae family. The virus utilizes cellular lipids and manipulates host lipid metabolism to ensure its replication, especially during virus invasion and replication steps. Therefore, identification of the molecular lipid metabolism pathways that are suitable targets is critical for the development of anti-CSFV therapeutics. In this study, we screened the anti-CSFV activity of 12 compounds targeting synthesis of cholesterol and fatty acids, cholesterol esters, and cholesterol transport. We found that 25-hydroxycholesterol (25HC), a regulator of cholesterol metabolism and transport, has potent anti-CSFV activity. Mechanistically, we showed that 25HC inhibited CSFV proliferation by blocking the entry of virions into porcine alveolar macrophages (3D4/21) by decreasing cholesterol abundance in the plasma membrane through activation of acyl-CoA:cholesterol acyltransferase (ACAT). Finally, we revealed that cholesterol 25-hydroxylase (CH25H), a redox enzyme that mediates 25HC production, also restricted CSFV infection via both enzyme activity-dependent and -independent mechanisms. Collectively, our results shed light on the mechanisms by which 25HC inhibits CSFV entry into cells and suggests a potential new therapeutic method against CSFV infection.

Kinesin-1 Regulates Endocytic Trafficking of Classical Swine Fever Virus along Acetylated Microtubules.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is an important and highly infectious pig disease worldwide. Kinesin-1, a molecular motor responsible for transporting cargo along the microtubule, has been demonstrated to be involved in the infections of diverse viruses. However, the role of kinesin-1 in the CSFV life cycle remains unknown. Here, we first found that Kif5B played a positive role in CSFV entry by knockdown or overexpression of Kif5B. Subsequently, we showed that Kif5B was associated with the endosomal and lysosomal trafficking of CSFV in the early stage of CSFV infection, which was reflected by the colocalization of Kif5B and Rab7, Rab11, or Lamp1. Interestingly, trichostatin A (TSA) treatment promoted CSFV proliferation, suggesting that microtubule acetylation facilitated CSFV endocytosis. The results of chemical inhibitors and RNA interference showed that Rac1 and Cdc42 induced microtubule acetylation after CSFV infection. Furthermore, confocal microscopy revealed that cooperation between Kif5B and dynein help CSFV particles move in both directions along microtubules. Collectively, our study shed light on the role of kinesin motor Kif5B in CSFV endocytic trafficking, indicating the dynein/kinesin-mediated bidirectional CSFV movement. The elucidation of this study provides the foundation for developing CSFV antiviral drugs. IMPORTANCE The minus end-directed cytoplasmic dynein and the plus end-directed kinesin-1 are the molecular motors that transport cargo on microtubules in intracellular trafficking, which plays a notable role in the life cycles of diverse viruses. Our previous studies have reported that the CSFV entry host cell is dependent on the microtubule-based motor dynein. However, little is known about the involvement of kinesin-1 in CSFV infection. Here, we revealed the critical role of kinesin-1 that regulated the viral endocytosis along acetylated microtubules induced by Cdc42 and Rac1 after CSFV entry. Mechanistically, once CSFV transported by dynein met an obstacle, it recruited kinesin-1 to move in reverse to the anchor position. This study extends the theoretical basis of intracellular transport of CSFV and provides a potential target for the control and treatment of CSFV infection.

The regulation of cell homeostasis and antiviral innate immunity by autophagy during classical swine fever virus infection.

CSFV (classical swine fever virus) is currently endemic in developing countries in Asia and has recently re-emerged in Japan. Under the pressure of natural selection pressure, CSFV keeps evolving to maintain its ecological niche in nature. CSFV has evolved mechanisms that induce immune depression, but its pathogenic mechanism is still unclear. In this study, using transcriptomics and metabolomics methods, we found that CSFV infection alters innate host immunity by activating the interferon pathway, inhibiting host inflammation, apoptosis, and remodelling host metabolism in porcine alveolar macrophages. Moreover, we revealed that autophagy could alter innate immunity and metabolism induced by CSFV infection. Enhanced autophagy further inhibited CSFV-induced RIG-I-IRF3 signal transduction axis and JAK-STAT signalling pathway and blocked type I interferon production while reducing autophagy inhibition of the NF-κB signalling pathway and apoptosis in CSFV infection cells. Furthermore, the level of CSFV infection-induced glycolysis and the content of lactate and pyruvate, as well as 3-phosphoglyceraldehyde, a derivative of glycolysis converted to serine, was altered by autophagy. We also found that silencing HK2 (hexokinase 2), the rate-limiting enzyme of glycolytic metabolism, could induce autophagy but reduce the interferon signalling pathway, NF-κB signalling pathway, and inhibition of apoptosis induced by CSFV infection. In addition, inhibited cellular autophagy by silencing ATG5 or using 3-Methyladenine, could backfill the inhibitory effect of silencing HK2 on the cellular interferon signalling pathway, NF-κB signalling pathway, and apoptosis.

Interaction of SERINC5 and IFITM1/2/3 regulates the autophagy-apoptosis-immune network under CSFV infection.

The host restriction factor serine incorporator 5 (SERINC5) plays a key role in inhibiting viral activity and has been shown to inhibit classical swine fever virus (CSFV) infection. However, the action of SERINC5 in the interaction between host cells and CSFV remains poorly understood. This study found that SERINC5 represses CSFV-induced autophagy through MAPK1/3-mTOR and AKT-mTOR signalling pathways. Further research showed that SERINC5 promotes apoptosis by repressing autophagy. Likewise, it was demonstrated that SERINC5 interacting proteins IFITM1/2/3 inhibit CSFV replication and regulate autophagy in a lysosomal-associated membrane protein LAMP1-dependent manner. In addition, IFITM1/2/3 interference promotes the NF-κB signalling pathway for potential immunoregulation by inhibiting autophagy. Finally, the functional silencing of IFITM1/2/3 genes was demonstrated to enhance the inhibitory effect of SERINC5 on autophagy. Taken together, These data uncover a novel mechanism through SERINC5 and its interacting proteins IFITM1/2/3, which mediates CSFV replication, and provides new avenues for controlling CSFV.

Valosin-containing protein (VCP/p97) is responsible for the endocytotic trafficking of classical swine fever virus.

Classical swine fever virus (CSFV), a member of the Flaviviridae enveloped RNA virus family, results in an epidemic disease that brings serious economic losses to the pig industry worldwide. Valosin-containing protein (VCP/p97), a multifunctional active protein in cells, is related to the life activities of many viruses. However, the role of VCP in CSFV infection remains unknown. In this study, it was first found that treatment of VCP inhibitors impaired CSFV propagation. Furthermore, overexpression or knockdown of VCP showed that it was essential for CSFV infection. Moreover, confocal microscopy and immunoprecipitation assay showed that VCP was recruited for intracellular transport from early endosomes to lysosomes. Importantly, knockdown of VCP prevented CSFV to release from early endosomes, suggesting that VCP is a key factor for CSFV trafficking. Taken together, our findings first demonstrate that the endocytosis of CSFV into PK-15 cells requires the participation of VCP, providing the alternative approach for the discovery of novel anti-flaviviridae drugs.

Cholesterol Biosynthesis Modulates CSFV Replication.

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) has resulted in severe losses to the pig industry worldwide. It has been proposed that lipid synthesis is essential for viral replication, and lipids are involved in viral protein maturation and envelope production. However, the specific crosstalk between CSFV and host cell lipid metabolism is still unknown. In this study, we found that CSFV infection increased intracellular cholesterol levels in PK-15 cells. Further analysis demonstrated that CSFV infection upregulated PCSK9 expression to block the uptake of exogenous cholesterol by LDLR and enhanced the cholesterol biosynthesis pathway, which disrupted the type I IFN response in PK-15 cells. Our findings provide new insight into the mechanisms underpinning the pathogenesis of CSFV and hint at methods for controlling the disease.

Rab22a cooperates with Rab5 and NS4B in classical swine fever virus entry process.

Classical swine fever virus (CSFV) is an ancient and economically important flavivirus that utilizes a Rab5-dependent endocytic pathway to enter host cells. Rab22a is a small GTPase that cooperates with Rab5 in the regulation of early endosome dynamics. Until now, the role of Rab22a in the flavivirus life cycle has been poorly defined. In this study, we systematically analyzed the role of Rab22a in CSFV proliferation and internalization using multiple viral replication analyses in combination with the overexpression, knockdown, and mutation of Rab22a, and found that Rab22a is involved in the entry process of CSFV. Confocal microscopy results showed that Rab22a colocalized with virus particles during the early phase of infection. Furthermore, by using glutathione S-transferase pull-down and co-immunoprecipitation assays, we verified the interaction between Rab22a and CSFV non-structural protein NS4B, and determined that NS4B can only bind to wild-type Rab22a, but not to the mutants Q64L and S19N. In addition, we explored the relationship between Rab22a, Rab5 and NS4B in CSFV internalization, and found out that these three proteins bind in early endosomes, and then through a Rab22a-Rab5-NS4B cascade allows the entry of CSFV. Taken together, our findings highlight the role of Rab proteins in CSFV internalization, and extend the understanding of the life cycle of flaviviruses.

ARF1 with Sec7 Domain-Dependent GBF1 Activates Coatomer Protein I To Support Classical Swine Fever Virus Entry.

Classical swine fever virus (CSFV), a positive-sense, enveloped RNA virus that belongs to the Flaviviridae family, hijacks cell host proteins for its own replication. We previously demonstrated that Golgi-specific brefeldin A (BFA) resistance factor 1 (GBF1), a regulator of intracellular transport, mediates CSFV infection. However, the molecular mechanism by which this protein regulates CSFV proliferation remains unelucidated. In this study, we constructed a series of plasmids expressing GBF1 truncation mutants to investigate their behavior during CSFV infection and found that GBF1 truncation mutants containing the Sec7 domain could rescue CSFV replication in BFA- and GCA (golgicide A)-treated swine umbilical vein endothelial cells (SUVECs), demonstrating that the effect of GBF1 on CSFV infection depended on the activity of guanine nucleotide exchange factor (GEF). Additionally, it was found that ADP ribosylation factors (ARFs), which are known to be activated by the Sec7 domain of GBF1, also regulated CSFV proliferation. Furthermore, we demonstrated that ARF1 is more important for CSFV infection than other ARF members with Sec7 domain dependence. Subsequent experiments established the function of coatomer protein I (COP I), a downstream effector of ARF1 which is also required for CSFV infection by mediating CSFV invasion. Mechanistically, inhibition of COP I function impaired CSFV invasion by inhibiting cholesterol transport to the plasma membrane and regulating virion transport from early to late endosomes. Collectively, our results suggest that ARF1, with domain-dependent GBF1 Sec7, activates COP I to facilitate CSFV entry into SUVECs. IMPORTANCE Classical swine fever (CSF), a highly contact-infectious disease caused by classical swine fever virus (CSFV) infecting domestic pigs or wild boars, has caused huge economic losses to the pig industry. Our previous studies have revealed that GBF1 and class I and II ARFs are required for CSFV proliferation. However, a direct functional link between GBF1, ARF1, and COP I and the mechanism of the GBF1-ARF1-COP I complex in CSFV infection are still poorly understood. Here, our data support a model in which COP I supports CSFV entry into SUVECs in two different ways, depending on the GBF1-ARF1 function. On the one hand, the GBF1-ARF1-COP I complex mediates cholesterol trafficking to the plasma membrane to support CSFV entry. On the other hand, the GBF1-ARF1-COP I complex mediates CSFV transport from early to late endosomes during the entry steps.

Classical swine fever virus NS4B protein interacts with MAVS and inhibits IL-8 expression in PAMs.

Classical swine fever virus (CSFV) infection causes a severe disease of pigs, resulting in significant economic losses. The CSFV NS4B protein is crucial for viral replication and pathogenicity. Interleukin 8 (IL-8), a main chemokine, is induced by multiple cell types and plays an essential role in host defense mechanisms against numerous viruses. It has been reported that NS4A of CSFV is involved in the induction of IL-8 expression in swine umbilical vein endothelial cells. However, the effect of CSFV NS4B on IL-8 expression is unknown. In this study, we showed that CSFV NS4B inhibited IL-8 expression in porcine alveolar macrophages (PAMs), and NS4B inhibited mitochondrial antiviral signaling protein (MAVS)-induced IL-8 expression. Moreover, CSFV NS4B interacted with MAVS. However, NS4B did not alter MAVS expression. Subsequently, we demonstrated that IRF3 knockdown or NF-κB inhibition reduced MAVS-induced IL-8 expression. Furthermore, the IRF3 and NF-κB pathways were activated by MAVS expression. However, CSFV NS4B inhibited MAVS-mediated NF-κB activation and IRF3 expression. Finally, CSFV NS4B inhibited IRF3 expression. Our findings reveal that CSFV NS4B interacts with MAVS and inhibits IL-8 expression by blocking the activation of IRF3 and NF-κB. Taken together, this study provides insights into the mechanism of NS4B-inhibited IL-8 expression.

Extracellular vesicles originating from autophagy mediate an antibody-resistant spread of classical swine fever virus in cell culture.

Free spread is a classical mode for mammalian virus transmission. However, the efficiency of this transmission approach is generally low as there are structural barriers or immunological surveillances in the extracellular environment under physiological conditions. In this study, we systematically analyzed the spreading of classical swine fever virus (CSFV) using multiple viral replication analysis in combination with antibody neutralization, transwell assay, and electron microscopy, and identified an extracellular vesicle (EV)-mediated spreading of CSFV in cell cultures. In this approach, intact CSFV virions are enclosed within EVs and transferred into uninfected cells with the movement of EVs, leading to an antibody-resistant infection of the virus. Using fractionation assays, immunostaining, and electron microscopy, we characterized the CSFV-containing EVs and demonstrated that the EVs originated from macroautophagy/autophagy. Taken together, our results showed a new spreading mechanism for CSFV and demonstrated that the EVs in CSFV spreading are closely related to autophagy. These findings shed light on the immune evasion mechanisms of CSFV transmission, as well as new functions of cellular vesicles in virus lifecycles.Abbreviations: 3-MA: 3-methyladenine; CCK-8: Cell Counting Kit-8; CSF: classical swine fever; CQ: chloroquine; CSFV: classical swine fever virus; DAPI, 4-,6-diamidino-2-phenylindole; EVs: extracellular vesicles; hpi: h post infection; IEM: immunoelectron microscopy; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MOI: multiplicity of infection; MVs: microvesicles; ND50: half neutralizing dose; PCR: polymerase chain reaction; PBS: phosphate-buffered saline; SEC: size-exclusion chromatography; siRNA: small interfering RNA; TEM: transmission electron microscopy.

Activation of Dendritic Cells in Tonsils Is Associated with CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus.

Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). The live attenuated C-strain vaccine is highly efficacious, initiating protection within several days of delivery. The vaccine strain is detected in the tonsil early after inoculation, yet little is known of the role that tonsillar immune cells might play in initiating protection. Comparing the C-strain vaccine with the pathogenic CSFV Alfort-187 strain, changes in the myeloid cell compartment of the tonsil were observed. CSFV infection led to the emergence of an additional CD163+CD14+ cell population, which showed the highest levels of Alfort-187 and C-strain infection. There was also an increase in both the frequency and activation status (as shown by increased MHC-II expression) of the tonsillar conventional dendritic cells 1 (cDC1) in pigs inoculated with the C-strain. Notably, the activation of cDC1 cells coincided in time with the induction of a local CSFV-specific IFN-γ+ CD8 T cell response in C-strain vaccinated pigs, but not in pigs that received Alfort-187. Moreover, the frequency of CSFV-specific IFN-γ+ CD8 T cells was inversely correlated to the viral load in the tonsils of individual animals. Accordingly, we hypothesise that the activation of cDC1 is key in initiating local CSFV-specific CD8 T cell responses which curtail early virus replication and dissemination.