B组柯萨奇病毒3Cpro切割AUF1蛋白引起心肌炎症损伤的机制研究

Coxsackievirus B (CVB) is the major pathogen of viral carditis, no effective drug or therapy against the infection is available. CVB 3C protease (3Cpro) plays an important role in the viral maturation and pathogenesis due to its specific recognizing and cleavage of viral precursor and multiple host proteins. RNA-binding protein AUF1 can bind to mRNA 3' untranslation region of multiple inflammatory cytokines, which may affect the stability of them. Previously, we found that AUF1 protein shuttled from nucleus to cytoplasm and its expression decreased significantly during the CVB infection. We hypothesize that it is probably that 3Cpro induces the abnormal expression of inflammatory cytokines through the AUF1 cleavage. The project aims to clarify the pathology of CVB3 infection-induced AUF1 cleavage: ① To determine the effect of 3Cpro on AUF1 cleavage and the cleavage sites in vitro;② To examine the impact of 3Cpro-induced AUF1 cleavage on inflammatory cytokines mRNA expression in vitro;③To clarify AUF1 cleavage in myocardia cells and the effect on inflammatory cytokines expression, and confirm that this is one of the mechanisms of CVB induced myocarditis. The study may provide a new target and strategy for anti-CVB infection.

B组柯萨奇病毒（CVB）是病毒性心肌炎主要病原，尚无有效治疗手段。CVB 3C蛋白酶（3Cpro）能特异识别并切割病毒前体蛋白及多种宿主蛋白，在病毒成熟及致病中起重要作用。RNA结合蛋白AUF1能识别多种炎性细胞因子mRNA 3'非编码区AU富集区，促进其降解。我们前期发现在CVB感染时AUF1有核→质转移且表达下调，并发现AUF1蛋白上存在3Cpro识别序列。据此推测3Cpro可能通过切割AUF1导致下游炎性细胞因子表达异常。本项目从三方面明确CVB切割AUF1的病理作用：①细胞实验确定3Cpro切割AUF1及确定切割位点；②细胞实验观察3Cpro切割AUF1以及对炎性细胞因子mRNA表达的影响；③明确CVB体内感染对心肌细胞AUF1切割及炎性细胞因子的表达异常的影响,以阐明AUF1切割是CVB诱发心肌炎症的重要机制之一。本项目为抗CVB感染的防止提供新靶点和策略。

B组柯萨奇病毒（CVB）是病毒性心肌炎主要病原，尚无有效治疗手段。CVB3 3C蛋白酶（3C proteasome，3Cpro）能特异性识别并切割病毒自身前体蛋白及多种宿主蛋白，在病毒成熟及致病中发挥重要作用。RNA结合蛋白AUF1能够识别多种炎性细胞因子mRNA 3’非编码区的AU富集区，促进或抑制mRNA降解。本研究发现：①CVB感染时，CVB3/CVB3 3Cpro通过识别特异性切割位点AATQ/G发挥切割AUF1的作用，并伴有AUF1核→质转移；②在CVB3感染的细胞中，发现AUF1可被募集重定位至胞质应激颗粒中； ③应激时RNA选择性多聚腺苷酸化倾向于利用短的3’UTR处的pA位点，且发现其位点前50nt处的顺式作用调控原件AAAA；④利用细胞因子蛋白芯片的方法检测CVB3切割AUF1对下游炎性细胞因子表达谱的影响，筛选显著差异基因进行验证，确定CVB3感染过程中AUF1蛋白水平变化对宿主炎性损伤的影响；⑤AUF1通过转录后调控PDL1蛋白的表达水平，阻断PDL1通路信号，诱导宿主细胞的免疫抑制作用；⑥CVB3感染切割AUF1时宿主宏观蛋白质组学变化；综上，已明确CVB体内感染对心肌细胞AUF1切割及炎性细胞因子的表达异常的影响,以阐明AUF1切割是诱发心肌炎症的重要机制之一，并且AUF1在整体转录组及蛋白组学调控过程中发挥重要作用。