牛病毒性腹泻病毒Erns蛋白激活NOD样受体热蛋白结构域相关蛋白3炎症小体诱发细胞焦亡的研究

Erns glycoprotein of bovine viral diarrhea virus activates NLRP3 inflammasome to induce pyroptosis

摘要:

【目的】牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)是引起牛病毒性腹泻-黏膜病的关键病毒。BVDV的结构蛋白Erns可在病毒感染的初期削弱宿主的免疫防御，引发牛群炎症反应。核苷酸寡聚化结构域样受体(nucleotide-binding oligomerization domain, NOD)热蛋白结构域相关蛋白3 (NLRP3)炎症小体是NOD样受体(NOD-like receptor, NLRs)家族重要成员，调控炎症性疾病的发生发展，同时激活的NLRP3炎症小体能够引起宿主细胞焦亡，进而诱发级联放大的炎症反应。但BVDV Erns蛋白在BVDV感染诱发炎症反应的分子机制尚不清楚。【方法】为进一步探索Erns蛋白对BVDV感染激活NLRP3炎症小体诱发细胞焦亡的影响，构建了BVDV Erns蛋白的真核表达质粒pCMV-HA-Erns，过表达BVDV Erns蛋白，检测BVDV感染细胞中NLRP3炎症小体组分[半胱氨酸蛋白酶(caspase-1)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein, ASC)和NLRP3]、IL-1β的mRNA转录水平和蛋白表达水平，以及细胞死亡调节蛋白(gasdermin D, GSDMD)的基因表达和蛋白剪切情况，并通过扫描电镜观察牛睾丸(bovine testis, BT)细胞膜成孔及BT细胞内容物释放情况，以分析Erns蛋白诱导BT细胞产生细胞焦亡。【结果】Erns蛋白能够显著引起NLRP3炎症小体活化进而激活caspase-1，活化的caspase-1一方面切割GSDMD，形成有活性的GSDMD-N端并在BT细胞膜形成孔洞，释放内容物，诱导BT细胞发生细胞焦亡；另一方面活化的caspase-1切割pro-IL-1β，形成有活性的IL-1β，并释放到BT细胞外，引起BT细胞上清中IL-1β水平上升。【结论】系统解析了BVDV Erns蛋白激活NLRP3炎症小体介导细胞焦亡的产生，对疫苗及治疗药物的研制具有重要指导意义。

Abstract:

[Objective] Bovine viral diarrhea virus (BVDV) is the key pathogen that causes bovine viral diarrhea-mucosal disease. The structural protein Erns of BVDV can weaken the host immune defense at the initial stage of virus infection and induce an inflammatory response in cattle. Nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome is an important member of the NLR family, regulating the occurrence and development of inflammatory diseases. The activated NLRP3 inflammasome can cause pyroptosis of host cells, thereby inducing cascading inflammatory responses. However, the molecular mechanism of the Ern protein in inducing an inflammatory response in BVDV infection remains unclear. [Methods] To explore the effect of Erns protein on the NLRP3 inflammasome-induced pyroptosis during BVDV infection, we constructed a eukaryotic expression plasmid pCMV-HA-Erns to overexpress the Ern protein of BVDV. The mRNA and protein levels of cysteine-dependent aspartate-specific protease 1 (caspase-1), apoptosis-associated speck-like protein containing CARD (ASC), NLRP3, and interleukin-1 beta (IL-1β) in BVDV-infected cells were determined. The gene expression and cleavage of gasdermin D (GSDMD) were examined. Furthermore, electron microscopy was employed to observe the bovine testis (BT) cells and examine the pyroptosis. [Results] The Erns protein significantly activated the NLRP3 inflammasome and caspase-1. The activated caspase-1 cleaved GSDMD to produce active GSDMD-N and create holes on the BT cell membrane for the contents releasing, thereby inducing pyroptosis. Furthermore, the activated caspase-1 cut pro-IL-1β to produce activated IL-1β in the BT cell supernatant. [Conclusion] We systematically analyze the role of BVDV Erns protein in activating NLRP3 inflammasome to mediate pyroptosis, which is vital for the development of vaccines and therapeutic drugs for the prevention of BVDV.