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

Huifang Lv,Kangkang Guo,Jie Wang,Wang Dong,Yanming Zhang,Zhi Cao,Xiaomeng Li,Gui Qian,Qizhuang Lv,Chengbao Wang

doi:10.1038/s41598-017-06934-1

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.

Highlights

Classical swine fever is a highly contagious viral disease caused by classical swine fever virus (CSFV), resulting in high mortality rates and causing tremendous economic losses in the swine industry
We verified 26 host-cell proteins as interacting with Classical swine fever virus (CSFV) non-structural protein 3 (NS3) according to yeast two-hybrid results, with the identified proteins predicted to be involved in metabolism, endocytosis, apoptosis, heat-shock response, and immune response according to bioinformatics analysis
tumour necrosis factor receptor-associated factor 6 (TRAF6) was verified as interacting with CSFV NS3 by co-IP and glutathione S-transferase (GST) pull-down assays

Summary

Introduction

Classical swine fever is a highly contagious viral disease caused by classical swine fever virus (CSFV), resulting in high mortality rates and causing tremendous economic losses in the swine industry. NS3 is an internal ribosome entry site (IRES)-binding protein that increases IRES-dependent translation; CSFV NS5A and NS5B can reduce NS3-IRES interactions by competitively binding to the same sites in IRES-containing RNA sequences. 2 1 1 1 1 functioning as an ubiquitin E3 ligase essential for activation of downstream signalling cascades. When ligand or stimulator, such as poly (I:C) or lipopolysaccharide (LPS), is added, TLR recruits adaptor proteins, including MyD88, TLR/IL-1R-domain containing adaptor inducing interferon-beta, and TRAF6. K63-polyubiquitination targets TRAF6, and ubiquitinated TRAF6 initiates signalling cascades[15, 16] that promote the rapid translocation of NF-κB into the nucleus, followed by phosphorylation of NF-κB p65 and transcriptional activation of various target genes, such as type 1 interferon (IFN) and inflammatory cytokines[17,18,19]. We hypothesized that TRAF6 might affect CSFV replication by regulating the NF-κB-signalling pathway

Methods

Results

Conclusion