猪圆环病毒2型通过内质网应激介导细胞凋亡的机制研究

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease with severe immunosuppression and can cause apoptosis. Our recent work indicated that the capsid protein (Cap) of PCV2 elicits endoplasmic reticulum (ER) stress and triggers apoptosis through activation of the PERK-CHOP pathway and release of calcium from ER in PK-15 cells with unknown mechanisms. The CHOP knockout PK-15 cell line will be constructed to investigate the roles and molecular mechanisms of CHOP in ER stress-mediated apoptosis during PCV2 infection. Gene silencing, chemical inhibition, adenoviral transduction, western blotting, quantitative PCR, fluorescence resonance energy transfer assay, flow cytometry and TUNEL approaches will be employed to study the relationship between CHOP-ERO1α and oxidative stress and ER calcium release in inducing apoptosis during PCV2 infection in PK-15 cells. In addition, full-length and truncated Cap will be constructed to identify the regions of Cap critical for inducing the cell responses. This will increase the knowledge of the connection between ER stress and apoptosis during DNA virus infection as well as shed light on the development of novel antiviral drugs targeting these pathways.

猪圆环病毒2型（PCV2）是引起猪免疫抑制相关疾病的重要病原并诱导宿主细胞发生凋亡。前期研究发现PCV2感染猪PK-15细胞后能通过病毒衣壳蛋白Cap激活PERK-CHOP通路，引起内质网钙离子外流并促进细凋亡，但机理不清。本项目将通过构建CHOP基因敲除PK-15细胞系研究CHOP在PCV2感染诱导细胞从内质网应激走向凋亡中的作用和分子机制。应用基因沉默、化学抑制、腺病毒表达、免疫印迹、荧光定量PCR、荧光能量共振转移法、流式细胞术和TUNEL等技术手段解析CHOP-ERO1α途径与氧化应激和内质网钙离子外流之间的联系，阐明PCV2通过CHOP诱导凋亡的内在机理。最后利用全长和截短Cap分析PCV2感染通过内质网应激诱导凋亡的关键病毒蛋白结构域。本研究对于揭示DNA病毒感染通过内质网应激诱导细胞凋亡的分子机理具有重要意义，为探索以内质网应激为靶向的PCV2抗病毒辅助治疗方案提供理论依据。

猪圆环病毒2型（PCV2）是导致断奶仔猪多系统衰竭综合征的病原，给养猪业造成严重的经济损失。PCV2感染可以诱导内质网应激和氧化应激，这些细胞反应与凋亡之间都存在联系。但在PCV2感染诱导的凋亡与内质网应激和氧化应激的联系机制并不明确。本研究证实PCV2感染能增强促凋亡蛋白CHOP和内质网氧化酶1α（ERO1α）的表达。通过siRNA抑制CHOP的表达或通过基因敲低或化学抑制剂抑制ERO1α的表达均能抑制PCV2感染诱导产生的ROS水平，Ca2+水平和凋亡率。过表达ERO1α则能增加PCV2感染诱导产生的ROS水平，Ca2+水平和凋亡率。使用ROS清除剂能减少PCV2感染诱导产生的ROS水平，Ca2+水平和凋亡率，但同时也减少了CHOP和ERO1α的蛋白表达水平。综上，我们认为PCV2感染通过CHOP-ERO1α-ROS信号通路诱导细胞凋亡。