SARS-CoV-2非结构蛋白Nsp5与Bcl2相互作用诱导线粒体损伤激活NLRP3炎症小体的分子机制研究

炎症因子失调是COVID-19病程迅速发展的重要原因之一，线粒体损伤在病毒感染引起炎症反应中发挥重要桥梁作用。临床分析发现新冠病毒感染引发宿主线粒体损伤和炎症因子增多，但具体分子机制尚不清楚。申请人前期发现新冠病毒非结构蛋白Nsp5能与Bcl2相互作用和上调线粒体上Bak蛋白水平；诱发线粒体损伤及线粒体DNA移位；同时促进NLRP3和IL-1β表达增多；抑制线粒体DNA则缓解Nsp5对IL-1β的诱导。因此推测：Nsp5通过干扰Bcl2-Bak相互作用诱导线粒体损伤，进而激活NLRP3炎症小体导致IL-1β表达增多。本项目拟运用质粒过表达、基因敲降及真病毒感染等手段，在细胞系和原代细胞上阐明Nsp5通过竞争性抑制Bcl2-Bak相互作用诱导线粒体损伤进而激活NLRP3炎症小体的分子机制，并确定其作用位点。本研究将揭示新冠病毒感染引发炎症因子失调新机制，为其药物干预提供新靶点和新线索。

The dysregulation of inflammatory factors is one of the important reasons for the rapid development of COVID-19. Mitochondrial damage plays an important role in the inflammatory response caused by viral infection. Clinical analysis showed that SARS-CoV-2 infection induced mitochondrial damage and increased inflammatory factors in the host, but the molecular mechanism remains unclear. Moreover, we discovered that non-structural protein 5 (Nsp5) of SARS-CoV-2 could interact with Bcl2 and promoted the expression of Bak on mitochondria. Meanwhile, Nsp5 could induce mitochondrial damage and mtDNA translocation. Further research found that Nsp5 promoted the increase of NLRP3 and IL-1β expression. What is more, inhibition of mtDNA alleviated the expression of IL-1β caused by Nsp5. Therefore, it can be proposed that Nsp5 induces mitochondrial damage by interfering with the Bcl2-Bak interaction, and then activates the NLRP3 inflammasome to increase the expression of IL-1β. In the present study, we will combine plasmid overexpression, gene knockdown, and virus infection to clarify the molecular mechanism that Nsp5 induces mitochondrial damage by competitively inhibiting the Bcl2-Bak interaction, and activates NLRP3 inflammasome to promote IL- 1β expression on cell lines and primary cells. This study will reveal a new mechanism of inflammatory factors dysregulation caused by SARS-CoV-2, and provide new targets and clues for COVID-19 treatment.