TRAF6 is a novel NS3-interacting protein that inhibits classical swine fever virus replication

Abstract

Classical swine fever virus (CSFV) non-structural protein 3 (NS3) is a multifunctional non-structural protein that plays a major role in viral replication. However, how exactly NS3 exerts these functions remains unknown. Here, we identified tumour necrosis factor receptor-associated factor 6 (TRAF6) as a novel NS3-interacting protein via yeast two-hybrid analysis, co-immunoprecipitation, and glutathione S-transferase pull-down assays. Furthermore, we observed that TRAF6 overexpression significantly inhibited CSFV replication, and TRAF6 knockdown promoted CSFV replication in porcine alveolar macrophages. Additionally, TRAF6 was degraded during CSFV infection or NS3 expression exclusively, indicating that CSFV and TRAF6 were mutually antagonistic and that TRAF6 degradation might contribute to persistent CSFV replication. Moreover, nuclear factor-kappa B (NF-κB) activity and interferon (IFN)-β and interleukin (IL)-6 expression were increased in TRAF6-overexpressing cells, whereas TRAF6-knockdown cells exhibited decreased NF-κB activity and IFN-β and IL-6 levels. Notably, TRAF6 overexpression did not reduce CSFV replication following inhibition of NF-κB activation by p65 knockdown. Our findings revealed that TRAF6 inhibits CSFV replication via activation of NF-κB-signalling pathways along with increases in the expression of its targets IFN-β and IL-6. This work addresses a novel aspect concerning the regulation of innate antiviral immune response during CSFV infection.

Classical Swine Fever Virus Infection and Its NS4A Protein Expression Induce IL-8 Production through MAVS Signaling Pathway in Swine Umbilical Vein Endothelial Cells

“…HEK293T cells are commonly used for production of lentivirus, since having the high transfection efficiency and producing high titers of lentivirus. The lentiviruses with MAVS knockdown were produced as previously described method (Lv et al, 2017a ). Briefly, HEK293T cells were co-transfected with the plasmids MAVS shRNAs (MAVS-sh1/2) and three other plasmids (pGag/Pol, pRev, pVSVG, conserved in our lab) using TurboFect (Thermo, USA).…”

“…Thus, ST cells are commonly used to detect the extracellular titers of CSFV (Liang et al, 2017 ; Lv et al, 2017b ). CSFV titers in culture supernatants were determined by IFA as the following methods (Lv et al, 2017a ). ST cells in 96-well plate were infected with the progeny viruses by gradient dilution.…”

mTORC1 Negatively Regulates the Replication of Classical Swine Fever Virus Through Autophagy and IRES-Dependent Translation

“…CSFV-NS5A promotes viral replication and spreading by interaction with heat shock protein 70 (HSP70) or by localization within the endoplasmic reticulum (ER) where it induces oxidative stress in vascular endothelial cells ( Zhang et al., 2015 , He et al., 2012 ). CSFV-NS5A can also inhibit internal ribosome entry site (IRES)-mediated translation by interaction with eukaryotic elongation factor 1A (eEF1A) ( Li et al., 2015a ), whereas CSFV-NS3 interacts with TRAF6 and degrades it to promote CSFV replication via the NF-κB signaling pathway ( Lv et al., 2017 ), and CSFV-N pro interacts with interferon regulatory factor 3 (IRF-3) or IRF-7 and blocks type I interferon induction ( Bauhofer et al., 2007 , Fiebach et al., 2011 ).…”

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

“…1 College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China. 2 Tianjin Animal Husbandry and Veterinary Research Institute, Tianjin, China. 3 Henan University of Animal Husbandry and Economy, Zhengzhou, China.…”

“…Classical swine fever (CSF), which is caused by the CSF virus (CSFV) of family Flaviviridae, is a highly contagious viral disease of pig and has serious socioeconomic implications [1]. CSFV is an enveloped RNA virus with positive-sense genomic RNA and an open reading frame that encodes a precursor polyprotein of 3898 amino acids that are co-and post-translationally processed into four structural proteins (C, E rns , E1, E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) [2].…”