猪繁殖与呼吸综合征病毒NSP1β与HSP40相互作用抑制I型干扰素的产生及其分子机制

Porcine reproductive and respiratory syndrome virus (PRRSV) NSP1β inhibits type I interferon production by antagonizing IRF3 phosphorylation and nuclear translocation. However, the molecular mechanism of IRF3 phosphorylation in PRRSV-mediated innate immune response is less understood. In previous studies, NSP1β was used as a "bait" to search for its novel interacting factors by using yeast two-hybrid system to screen porcine alveolar macrophages cDNA library. We found that NSP1β interacts with heat shock protein (HSP40) and the interaction was confirmed in mammalian cells by co-immunoprecipitation and co-localization. Considering the known function of HSP40 in regulation of IRF3 phosphorylation during other virus infection, we proposed a hypothesized that PRRSV NSP1β exploits HSP40 to inhibit IRF3 phosphorylation and type I interferon production. In the project, we will test the hypothesis and elucidate the molecular mechanism of IRF3 phosphorylation. 1) We will confirm the interaction of NSP1β and HSP40 in PAM post PRRSV infection by co-immunoprecipitation and co-localization. 2) We will confirm the release P58IPK from HSP40-P58IPK complex by NSP1β competitive binding to HSP40. 3) We will investigate whether NSP1β regulates type I IFN through HSP40 by overexpression and knock-down. 4) We will identify the possible signaling pathway of HSP40 in regulation of IRF3 phosphorylation. 5) We will analyse the expression and phosphorylation difference of each node in the pathway by overexpression and knock-down. 6) At last we will investigate type I interferon production and virus replication. This project is expected to uncover the molecular mechanism of inhibiting type I interferon production by the interaction of NSP1β and HSP40, providing useful data for understanding the mechanism of inhibition type I interferon production in PRRSV-infected cells.

猪繁殖与呼吸综合征病毒（PRRSV）非结构蛋白1β（NSP1β）通过调控干扰素调节因子3（IRF3）的磷酸化而抑制I型干扰素产生，但具体机制尚不清楚。本研究前期以PRRSV NSP1β为"诱饵"，通过酵母双杂交筛选猪肺泡巨噬细胞文库，发现并证实NSP1β与热休克蛋白40（HSP40）相互作用。已有研究报道HSP40在其它病毒蛋白抑制IRF3磷酸化过程中发挥作用，因此，本研究将通过免疫共沉淀、共定位、上调表达和下调表达等方法研究HSP40在NSP1β抑制I型干扰素产生中的功能；通过研究HSP40作用通路中重要分子的表达及磷酸化水平的变化，以及对I型干扰素产生和病毒复制水平的影响，揭示NSP1β与HSP40相互作用抑制宿主I型干扰素产生的分子机制，研究结果将为阐明PRRSV抑制I型干扰素产生的分子机制提供科学依据。

猪繁殖与呼吸综合征(PRRS)由猪繁殖与呼吸综合征病毒(PRRSV)引起的一类严重接触性传染病。PRRSV感染引起严重的肺部炎症症状，在感染早期就可以检测到大量的促炎性细胞因子。高致病毒株HuN4和SDA2均能诱导这些细胞因子的高水平表达和分泌，进一步探索发现PRRSV感染可以诱导NLRP3炎症小体复合物的形成。本研究深入探索激活炎症小体的其它病毒组分，结果发现PRRSV基因组RNA可以诱导较高水平的IL-1β分泌。本研究设计了从PAM细胞中钓取RNA受体的筛选模型，通过免疫共沉淀试验和串联质谱分析筛出了DDX19A，并发现DDX19A只特异地与NLRP3炎症小体复合物中的NLRP3相互作用，而不与ASC和caspase-1相互作用。本研究进一步证明DDX19A能直接结合PRRSV基因组RNA及其5’UTR和3’UTR转录物。通过慢病毒介导敲低DDX19A的表达显著抑制PRRSV基因组RNA及其5’UTR转录物和细菌RNA诱导的NLRP3炎症小体活化和IL-1β分泌，但不影响poly I:C和LPS/ATP诱导NLRP3炎症小体依赖的IL-1β分泌。最后，本研究发现敲低DDX19A的表达后显著抑制PRRSV在PAM细胞和Marc-145细胞中的复制水平。.综上所述，本研究深入地从分子机制水平揭示了PRRSV感染诱导强烈炎症反应的机理，明确指出了诱导IL-1β分泌的PRRSV组分及信号传导通路，证明PRRSV基因组RNA和细菌RNA均可以通过新鉴定的RNA受体DDX19A激活NLRP3炎症小体，诱导炎症反应，从理论上解释了细菌继发感染在PRRSV感染过程中的致病机理，为防控PRRS和开发抗炎药物提供重要参考。同时，还证明了DDX19A对病毒复制过程的调控作用。