Npro蛋白可抑制poly(I:C)和流感病毒激活的天然免疫应答（英文）

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

摘要:

【目的】猪瘟病毒(classical swine fever virus，CSFV)感染猪引起出血综合征和免疫抑制，是一种高度传染性的猪病。本研究以CSFV为研究对象，探究CSFV抑制宿主天然免疫的机制及其在免疫抑制条件下对甲型流感病毒(influenza A virus，IAV)感染的潜在作用。【方法】首先，为探究CSFV对宿主天然免疫的影响，利用反转录PCR(reverse transcription-PCR，RT-PCR)、荧光定量PCR(reverse transcription-quantitative PCR，RT-qPCR)和Western blotting技术检测PK-15细胞感染CSFV后对Poly(I:C)诱导干扰素(interferons，IFNs)、干扰素诱导基因(IFN-stimulated genes，ISGs)和信号传导子和转录活化子1(signal transducer and activator of transcription 1，STAT1)磷酸化的影响。其次，利用过表达CSFV蛋白的细胞系筛选并确定抑制天然免疫的关键蛋白。最后，在过表达CSFV N^pro蛋白的细胞系上，利用RT-PCR、RT-qPCR、Western blotting和病毒噬斑实验技术研究N^pro蛋白对天然免疫和IAV感染的影响。【结果】CSFV可抑制poly(I:C)诱导的Ⅰ型和Ⅲ型IFN的表达。CSFV N^pro蛋白在体外直接抑制STAT1的磷酸化，引起寡腺苷酸合成酶样蛋白(OASL)、2',5'-寡腺苷酸合成酶1(OAS1)、干扰素诱导跨膜蛋白3(IFITM3)和干扰素刺激基因15(ISG15)表达下调。重要的是，CSFV N^pro蛋白可抑制IAV诱导的Ⅰ型和Ⅲ型IFN的表达，并导致STAT1的磷酸化和OASL、OAS1、IFITM3和ISG15的表达显著下降，进而显著促进IAV在过表达N^pro蛋白的PK-15细胞中复制。【结论】猪瘟病毒的N^pro蛋白能够拮抗由poly(I:C)和IAV所激活的天然免疫应答，表明N^pro蛋白可抑制RIG-I依赖的信号通路并可促进IAV在表达N^pro蛋白的PK15细胞中复制。

关键词:猪瘟病毒;甲型流感病毒;N^pro;天然免疫;干扰素;信号传导子和转录活化子1 (STAT1)

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.

Key words:classical swine fever virus (CSFV);influenza A virus (IAV);N^pro;innate immunity;interferon;signal transducer and activator of transcription 1