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.

RPS3-induced antiviral cytokines inhibit the proliferation of classical swine fever virus.

Classical swine fever virus (CSFV) infection results in serious economic losses to the pig industry. This positive-sense RNA virus hijacks cell host proteins for its own replication. Although previous studies have shown that RPS3, a 40S ribosomal subunit protein, is mainly required for DNA repair, apoptosis and inflammation, the effect of RPS3 on CSFV replication remains uncertain. Thus, we investigated the potential role of RPS3 in CSFV infection in RPS3-knockdown and -overexpressing cell lines using real-time fluorescence quantitative PCR (RT-qPCR) and indirect immunofluorescence assays. Results showed that knockdown of RPS3 by lentiviruses enhanced CSFV replication, whereas overexpression of RPS3 by lentiviruses inhibited CSFV replication. These findings indicated the antiviral role of RPS3 in CSFV infection. Subsequent experiments revealed that CSFV replication was inhibited in cells cultured with the supernatants of RPS3-overexpressing cell, suggesting that the RPS3-mediated inflammatory response was involved in CSFV infection. Furthermore, enzyme-linked immunosorbent assay (ELISA) revealed that the secretion of antiviral cytokines (IL-8 and INF-ß) was increased in cells with sufficient RPS3 expression but decreased in cells lacking RPS3 expression. RT-qPCR and immunofluorescence assays revealed that CSFV infection inhibited RPS3-mediated antiviral cytokine secretion. Taken together, these findings reveal that RPS3 is a novel antiviral factor that inhibits CSFV proliferation by increasing antiviral cytokine secretion. Keywords: classical swine fever virus; ribosome protein S3; IL-8; INF-ß.

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.

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.

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.

Rab18 binds to classical swine fever virus NS5A and mediates viral replication and assembly in swine umbilical vein endothelial cells.

Classical swine fever virus (CSFV), a positive-sense RNA virus, hijacks cell host proteins for its own replication. Rab18, a small Rab GTPase, regulates intracellular membrane-trafficking events between various compartments in cells and is involved in the life cycle of multiple viruses. However, the effect of Rab18 on the production of CSFV remains uncertain. In this study, we showed that knockdown of Rab18 by lentiviruses inhibited CSFV production, while overexpression of Rab18 by lentiviruses enhanced CSFV production. Subsequent experiments revealed that the negative-mutant Rab18-S22 N inhibited CSFV infection, while the positive-mutant Rab18-Q67 L enhanced CSFV infection. Furthermore, we showed that CSFV RNA replication and virion assembly, measured by real-time fluorescence quantitative PCR (RT-qPCR), indirect immunofluorescence assay (IFA), and confocal microscopy, were reduced in cells lacking Rab18 expression. In addition, co-immunoprecipitation, GST-pulldown, and confocal microscopy assays revealed that Rab18 bound to the viral protein NS5A. Further, NS5A was shown to be redistributed in Rab18 knockdown cells. Taken together, these findings demonstrate Rab18 as a novel host factor required for CSFV RNA replication and particle assembly by interaction with the viral protein NS5A.

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.