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.

Attenuation of nitric oxide bioavailability in porcine aortic endothelial cells by classical swine fever virus.

Classical swine fever (CSF) causes severe disease in pigs, characterized by hemorrhage, fever, and leucopenia. A primary target of the virus is endothelial cells, where a pro-inflammatory and pro-coagulant response occurs with downregulation of gap junctional communication; these changes establish a basis for haemostatic imbalance. The aim of this study was to gain an understanding of the effect of classical swine fever virus (CSFV) on endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability. Porcine aortic endothelial cells (PAECs) were infected with CSFV at different multiplicity of infection (M.O.I.) for 48 h. Downregulation of the transcription and translation levels of eNOS was detected by semi-quantitative RT-PCR, immunoconfocal microscopy, and western blotting. This was accompanied by a reduction in NO bioavailability and attenuation of angiogenesis. Without influence from the progeny virus titer, the decrease in eNOS protein was reversed by an ERK inhibitor (PD98059) and two PI3/Akt inhibitors (LY294002 and wortmannin). In addition, we found that the transcription factors AP1, Sp1, and GATA1/2 may be involved in the downregulation of eNOS promoter activity. In conclusion, infection of PAECs with CSFV attenuated the expression of eNOS and reduced NO bioavailability through activation of the ERK and PI3/Akt pathways.

Pathogenesis of granulocytopenia and bone marrow atrophy during classical swine fever involves apoptosis and necrosis of uninfected cells.

Granulocytopenia, a hematological hallmark of classical swine fever, is partially responsible for the suppression of innate immune defenses during classical swine fever. The present report demonstrates that this depletion was apparent as early as 3 days postinfection (p.i.). Both mature peripheral and bone marrow neutrophils were affected, whereas immature neutrophils increased absolutely in the periphery and coincidentally immature myeloid progenitors in the bone marrow. These data suggest that a pathogenic relationship exists between these compartments. The central event was not the arrest of hematopoietic cell proliferation or of the mobilization process, but instead apoptosis and possibly also necrosis were shown to play a role. This increase in apoptotic and dead cells was detected as early as 1-3 days p.i. In contrast, viral RNA in bone marrow hematopoietic cells (BMHC) was first detected 5 days p.i., and significant amounts of infected BMHC were detected only 7 days p.i., with the major target being the myeloid compartment. The increased caspase-3 activity observed supported a role for apoptotic cell death. Furthermore, the elevated caspase-9 activity indicated the involvement of the mitochondrial apoptotic pathway. Taken together, the results demonstrate that granulocytopenia and bone marrow atrophy are mediated by hematopoietic cell death and that indirect virus-host-mediated mechanisms are likely to be responsible.

[Activation of proteinases and acid phosphatase in swine leukocytes infected with the swine classical plague virus]. / Aktivatsiia proteinaz i kisloi fosfatazy v leikotsitakh svin'i, infitsirovannykh virusom klassicheskoi chumy svinei.

The activities of proteinases active at neutral pH and of acid phosphatase increases after freezing and defrosting and hypotonic shock in porcine leukocytes in vitro infected with the classical hog cholera virus in comparison with intact cells. The enzymes in the cells from animals both immune and nonimmune to hog cholera were activated upon infection with both virulent and vaccine strains of the virus. No proteinase activity was detected in the culture medium in which infected porcine leukocytes were cultured. Possible pathological reactions of a cell to viral infection are discussed.

[Activity of several serum enzymes in swine with acute plague]. / Prouchvane aktivnostta na niakoi serumni enzimi pri svine, bolni ot akutna chuma

Nonvaccinated pigs were infected with a pathogenic virus of swine fever in order to follow up the changes with some enzymes in their blood serum. It was found that there is a dependable rise in the values of the blood serum GOT (2.6.1. 1.) - From about 38 to approximately 108 mU per cu. cm; GPT (2. 6; 1.2.;-From about 12 up to 66 mU/cu. cm; LAP (3. 4. 1. 1)-from about 13 to 27 mU/cu. cm; and ALD (4. 1; 2. 7.) - from about 6 up to 19 mU/cu. cm. These changes were noticed as early as the 48th hour of infection, prior to the manifestation of the clinical symptoms of swine fever. They remained stable up to the end of the infection process. The activity of LDH (1. 1. 2. 7.) and amylase (3. 2. 1.1 .) was said to rise (not dependable statistically), and that of cholinesterase (3. 1 1. 8) - to drop gradually with the development of the infection process. The two phosphomonoesterases (3. 1. 3. 1 and 3. 1. 3. 2.) showed no essential changes. The changes taking place in the activity of these enzymes did not depend on the extent of the hemorrhagic diathesis characteristic of swine fever. So, this test, which has been unknown so far, is referred to as a characteristic paraclinical symptom of swine fever.