title: Thioredoxin 2 Is a Novel E2-Interacting Protein That Inhibits the Replication of Classical Swine Fever Virus.
abstract: The E2 protein of classical swine fever virus (CSFV) is an envelope glycoprotein that is involved in virus attachment and entry. To date, the E2-interacting cellular proteins and their involvement in viral replication have been poorly documented. In this study, thioredoxin 2 (Trx2) was identified to be a novel E2-interacting partner using yeast two-hybrid screening from a porcine macrophage cDNA library. Trx2 is a mitochondrion-associated protein that participates in diverse cellular events. The Trx2-E2 interaction was further confirmed by glutathione S-transferase (GST) pulldown, in situ proximity ligation, and laser confocal assays. The thioredoxin domain of Trx2 and the asparagine at position 37 (N37) in the E2 protein were shown to be critical for the interaction. Silencing of the Trx2 expression in PK-15 cells by small interfering RNAs significantly promotes CSFV replication, and conversely, overexpression of Trx2 markedly inhibits viral replication of the wild-type (wt) CSFV and to a greater extent that of the CSFV N37D mutant, which is defective in binding Trx2. The wt CSFV but not the CSFV N37D mutant was shown to reduce the Trx2 protein expression in PK-15 cells. Furthermore, we demonstrated that Trx2 increases nuclear factor kappa B (NF-κB) promoter activity by promoting the nuclear translocation of the p65 subunit of NF-κB. Notably, activation of the NF-κB signaling pathway induced by tumor necrosis factor alpha (TNF-α) significantly inhibits CSFV replication in PK-15 cells, whereas blocking the NF-κB activation in Trx2-overexpressing cells no longer suppresses CSFV replication. Taken together, our findings reveal that Trx2 inhibits CSFV replication via the NF-κB signaling pathway.
  
  
  OBJECTIVE
  Thioredoxin 2 (Trx2) is a mitochondrion-associated protein that participates in diverse cellular events, such as antioxidative and antiapoptotic processes and the modulation of transcription factors. However, little is known about the involvement of Trx2 in viral replication. Here, we investigated, for the first time, the role of Trx2 in the replication of classical swine fever virus (CSFV), a devastating pestivirus of pigs. By knockdown and overexpression, we showed that Trx2 negatively regulates CSFV replication. Notably, we demonstrated that Trx2 inhibits CSFV replication by promoting the nuclear translocation of the p65 subunit of NF-κB, a key regulator of the host's innate immunity and inflammatory response. Our findings reveal a novel role of Trx2 in the host's antiviral response and provide new insights into the complex mechanisms by which CSFV interacts with the host cell.
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Su
authors.initials: S
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Jinghan
authors.initials: J
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Wen-Rui
authors.initials: WR
authors.lastname: He
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Yongfeng
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Yajin
authors.initials: Y
authors.lastname: Liao
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Hua-Yang
authors.initials: HY
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Lian-Feng
authors.initials: LF
authors.lastname: Li
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Dong
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.
authors.forename: Yuzi
authors.initials: Y
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China huajiqiu@hvri.ac.cn.
authors.forename: Hua-Ji
authors.initials: HJ
authors.lastname: Qiu
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006692:Classical Swine Fever Virus / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D015723:Gene Library; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000378:metabolism; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013552:Swine; D013879:Thioredoxins / Q000378:metabolism / Q000494:pharmacology*; D020798:Two-Hybrid System Techniques; D014759:Viral Envelope Proteins / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology*
chemical_list: D017931:DNA Primers; D016328:NF-kappa B; D014759:Viral Envelope Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013879:Thioredoxins