Porcine Viperin protein inhibits the replication of classical swine fever virus (CSFV) in vitro

Wenliang Li, Li Mao, Yongguo Cao, Bin Zhou, Leilei Yang, Linxiao Han, Fei Hao, Tao Lin, Wenwen Zhang, Jieyuan Jiang

Classical swine fever virus (CSFV) is the causative pathogen of Classical swine fever (CSF), a highly contagious disease of swine. Viperin is one of the hundreds of interferon-stimulated genes (ISGs), and possesses a wide range of antiviral activities. The aim of this study was to explore whether porcine Viperin has the anti-CSFV activity. The influences of CSFV infection on Viperin expression and Newcastle disease virus (NDV)/Pseudorabies virus (PRV)-induced Viperin expression were examined in 3D4/21 cells and porcine peripheral blood mononuclear cells (PBMCs). Porcine Viperin gene was amplified to generate cell line PK-Vi over-expressing Viperin. CSFV was inoculated in the cell lines and viral load was detected by qRT-PCR, virus titration and Western blot. The influence of Viperin expression on CSFV binding, entry and release in the cells was also examined. The co-localization of Viperin with CSFV and its proteins (E2, NS5B) was determined by confocal laser scanning microscopy test. Co-IP assay was performed to check the interaction of Viperin with CSFV proteins. CSFV infection could not induce Viperin expression in vitro while significantly inhibiting NDV/PRV-induced Viperin expression at 12, 24 and 48 h post infection (hpi; P < 0.05). The proliferation of CSFV in PK-Vi was significantly inhibited at 24, 48 and 72 hpi (P < 0.05), comparing with control cells (PK-C1 expressing EGFP). Virus in both cell culture supernatants and cell pellets were reduced equally. CSFV binding and entry in the cells were not interfered by Viperin expression. These results indicated its anti-CSFV function occurred during the genome and/or protein synthesis step. Confocal laser scanning microscopy test showed the Viperin-EGFP protein co-localized with CSFV E2 protein in CSFV infected PK-Vi cells. Further experiments indicated that Viperin protein co-localized with E2 and NS5B proteins of CSFV in the transfected 293 T cells. Furthermore, Co-IP assay confirmed the interaction of Viperin with E2 protein, but not NS5B. Porcine Viperin effectively inhibited CSFV replication in vitro, potentially via the interaction of Viperin with CSFV E2 protein in cytoplasm. The results provided foundation for further studies of the interaction of Viperin with CSFV and other viruses.