猪流行性腹泻病毒nsp1蛋白对Ⅰ型干扰素应答的影响

Effect of porcine epidemic diarrhea virus nsp1 on type Ⅰ interferon response

摘要:

猪流行性腹泻病毒 (PEDV) 能抑制宿主Ⅰ型干扰素及其诱导的细胞抗病毒免疫应答，但是PEDV抑制Ⅰ型干扰素应答的分子机制尚不明了，尤其是PEDV非结构蛋白 (Nonstructural proteins，nsps) 在Ⅰ型干扰素应答中的调控作用研究不多。为研究PEDV非结构蛋白1 (nsp1) 对细胞Ⅰ型干扰素应答的影响，构建了真核表达载体pCAGGS-nsp1，采用Western blotting和间接免疫荧光试验确定nsp1在细胞中的表达。通过报告基因法、ELISA以及病毒复制抑制试验评估nsp1对Ⅰ型IFN的影响。结果显示，nsp1在转染细胞和病毒感染细胞中均高效表达；双荧光报告基因试验结果表明，nsp1能显著抑制IFN-β启动子活性，且具有剂量依赖性。ELISA结果显示，nsp1能显著抑制IFN-β蛋白的表达。水泡性口炎病毒 (VSV) 复制抑制试验结果显示，nsp1明显抑制poly(I:C)介导的Ⅰ型IFN的抗病毒作用。结果提示，nsp1作为PEDV的保守蛋白，具有拮抗Ⅰ型干扰素启动子活性和应答的功能，为揭示PEDV逃逸宿主天然免疫应答的机制和研发新型高效抗PEDV疫苗奠定基础。

Abstract:

Porcine epidemic diarrhea virus (PEDV) inhibits the host typeⅠinterferon and cellular antiviral response, but its inhibition mechanism is unclear, and the roles of PEDV nonstructural proteins in regulating typeⅠinterferon responses have been seldom studied. To study the effect of nsp1 on typeⅠinterferon response, nsp1 gene was cloned into a eukaryotic expression vector pCAGGS. The expression of nsp1 in transfected cells was determined by Western blot and indirect immunofluorescence assay. The effects of nsp1 on the induction of typeⅠinterferon were evaluated by dual luciferase reporter gene assay, ELISA and VSV bioassay. Western blot and indirect immunofluorescence assay showed that nsp1 was highly expressed in transfected cells and PEDV-infected cells. Dual luciferase reporter gene assay results indicated that nsp1 strongly inhibited the IFN-β promoter activity, and the inhibitory effect was nsp1 dose-dependent. ELISA results showed that nsp1 significantly inhibited the expression of IFN-β in protein level. And VSV replication-inhibition bioassay revealed that nsp1 significantly inhibited typeⅠIFN antiviral activities induced by poly(I:C). Our results implied that nsp1 was a highly conserved protein of PEDV and exhibited antagonistic function on interferon promoter activity. The results have laid a foundation for further understanding the immune evasion mechanism of PEDV and for developing new effective vaccine against PEDV.