人星状病毒非结构蛋白nsP1a C末端结构域致宿主细胞凋亡的机制

Human astroviruses（HAstV） are important pathogens that cause acute viral diarrhea in infants. Little is known about the mechanisms of astrovirus-induced diarrhea. Several lines of evidence has demonstrated that intestinal epithelial cells apoptosis plays a critical role in the events leading to diarrhea. Unfortunately, whether human astrovirus could induce apoptosis in intestinal epithelial cells was not examined. Caco2 cell derived from human colon carcinoma cell, which morphologically and functionally resembles the enterocytes lining the small intestine, is widely used as an in vitro model of the human small intestinal mucosa. We found that HAstV-I can induce Caco2 apoptosis. To further explore the mechanism of HAstV-I induced apoptosis of Caco2, we transfected HAstV-I non-structural protein nsP1a, nsP1b and coat protein ORF2 recombinants with GFP tag into Caco2 cells, respectively. The results revealed that nsP1a can induce apoptosis, however nsP1b and ORF2 can't induce apoptosis, suggesting nsP1a is involved in HAstV-I induced apoptosis. We further transfected the mutant in which nsP1a/4 was deleted into Caco2 cells, we found this mutant can't induce apoptosis of Caco2.Based on these data, we proposed that nsP1a/4 protein plays a critical role in HAstV-induced apoptosis. But the cellular and molecule mechanism remains unclear. In this project, we will (1) explore the underlying mechanism of nsP1a/4 in apoptosis of Caco2 and elucidate the signal pathway in this process, and the role of mitochondria pathway. (2) Screen and identify the amino acid sequence of nsP1a/4 which is directly involved in HAstV-induced apoptosis.nsP1a/4 protein different truncated forms recombinants with GFP tag were transfect into Caco2 and cell apoptosis were analyzed. (3) Identify the bind partners in Caco2 cells which are involved in nsP1a/4 inducing apoptosis. Detection of protein-protein interactions using the GST fusion protein pull-down technique and mass spectrometry analysis. Verify the function of the identified protein by biological experiments. The project will elucidate the cellular and molecule mechanisms of nsP1a/4 protein induce apoptosis from the view of host cell with the interaction of HAstV. It will play an important role to gain further insights into the pathogenic mechanism of human astrovirus and provide new biological evidence for the prevention and cure of HAstV-induced diarrhea.

人星状病毒（Human astrovirus，HAstV）是导致婴幼儿腹泻的重要病原体，但HAstV致腹泻的原因尚不明确。目前认为，肠上皮细胞凋亡是诱发腹泻的主要原因之一。课题组前期研究显示，HAstV I 能够诱导人结肠癌细胞Caco2凋亡，而病毒非结构蛋白nsP1a在诱导凋亡中起关键作用。进一步研究证实，凋亡作用是通过nsP1a 蛋白C末端（nsP1a/4）实现的，删除nsP1a/4后nsP1a不能再诱导凋亡。但nsP1a/4诱导凋亡的具体分子机制尚不清楚。本项目在前期工作的基础上，从nsP1a/4蛋白和细胞相互作用的角度出发，深入探讨nsP1a/4诱导凋亡的分子机制，进而鉴定促凋亡作用的关键氨基酸。采用GST-pulldwon和免疫共沉淀技术对Caco2细胞蛋白与nsP1a/4蛋白的相互作用进行初步探讨，从而为阐明HAstV致病机理奠定基础，同时也为防治HAstV腹泻提供新的线索。

人星状病毒（Human astrovirus，HAstV）是导致婴幼儿腹泻的重要病原体，但HAstV致腹泻的原因尚不明确。本研究从星状病毒非结构蛋白nsP1a 和细胞相互作用的角度出发，采用分子生物学、荧光共聚焦、流式细胞术、免疫印迹、酵母双杂交、GST-pull down、免疫共沉淀、基因沉默等技术，首次发现：（1）星状病毒能够诱导不同的宿主细胞产生凋亡。其中nsP1a 蛋白C末端蛋白nsP1a/4在宿主细胞凋亡过程中起到主要作用，其诱导凋亡的关键结构域在nsP1a/4蛋白的53-174aa处。（2）成功构建星状病毒宿主细胞Caco-2全长cDNA文库，采用酵母双杂交技术，在宿主细胞cDNA文库中筛选到与星状病毒非结构蛋白nsP1a互作的13种蛋白质；（3）宿主蛋白CD63与病毒感染宿主细胞密切相关。GST/His pull down、CoIP、荧光共聚焦等实验揭示nsP1a/4蛋白与CD63蛋白能够在体内外相互作用。（4）CD63的高表达会促进星状病毒病毒的感染与复制。CD63消减表达后，星状病毒的感染滴度下降。上述实验结果证实了本研究中有关“ 人星状病毒非结构蛋白nsP1a C末端结构域DD（53-174aa）能够诱导宿主细胞凋亡”的假说，为深入研究星状病毒的致病机制奠定基础，同时也为下一步开展星状病毒与宿主相互作用的分子机制提供了参考。