CSFV Npro protein can suppress innate antiviral responses induced by poly(I:C) and influenza A virus

Abstract:

[Objective] Classical swine fever virus (CSFV) causes a highly contagious porcine disease that is characterized by hemorrhage syndrome and immunosuppression. In this paper, we studied the mechanism of CSFV inhibiting the host's innate immunity and its potential effect on influenza A virus (IAV) infection under immunosuppressive conditions. [Methods] First, the expression of interferons (IFNs) and IFN-stimulated genes (ISGs) and the phosphorylation of signal transducer and activator of transcription 1 (STAT1) induced by poly(I:C) in PK-15 cells which had been infected by CSFV were detected by reverse transcription-PCR (RT-PCR), reverse transcription-quantitative PCR (RT-qPCR), and Western blotting to explore the effect of CSFV on host innate immunity. Then, cells overexpressing CSFV proteins were used to screen and identify the key protein inhibiting the innate immunity. Finally, the potential influence of CSFV N^pro on innate immunity and IAV infection was examined in N^pro-overexpressing PK-15 cells by RT-PCR, RT-qPCR, Western blotting, and plaque assay. [Results] CSFV suppressed expression of type Ⅰ and type Ⅲ IFNs induced by poly(I:C). CSFV N^pro protein directly inhibited STAT1 phosphorylation and caused down-regulation of oligoadenylate synthase-like (OASL) protein, 2'-5'-oligoadenylate synthase-1 (OAS1), IFN-induced transmembrane protein 3 (IFITM3), and interferon-stimulated gene 15 (ISG15) in vitro. Moreover, CSFV N^pro protein suppressed the expression of type Ⅰ and type Ⅲ IFNs induced by IAV, and caused a dramatic decline in phosphorylation of STAT1 and expression of OASL, OAS1, IFITM3, and ISG15, thereby significantly promoting the replication of IAV in the N^pro-overexpressing PK-15 cells. [Conclusion] CSFV N^pro protein antagonizes the innate antiviral response induced by poly(I:C) and IAV, suggesting that N^pro protein can inhibit the RIG-I-dependent signaling pathway and promote IAV replication in the N^pro-expressing PK-15 cells.