猪圆环病毒感染细胞自噬与凋亡调控的信号传导机制

Porcine circovirus 2 is the causative agent of post-weaning wasting syndrome that incurs great economic loss to the pig industry. In addition to inducing apoptosis, we have also found that PCV2 explores the autophagic machinerary for enhanced replication in PK-15 cells. However, it remains largely unknown on the mechanisms of PCV2-induced lymphoid depletion in affected pigs. We plan to make systemic approaches on (i) the involvement of endoreticulum stress, autophagy or apoptosis in PCV2-infected cells and the underlying signal pathways; (ii) the major domain of the viral proteins causing such cellular response(s); (iii) the relationship of such cellular responses in cultured cells and in mice that are infected by recombinant PCV2 carrying mutations on the putative protein domain(s) as compared to the wild-type virus; and (iv) the relationship between cellular responses and virus replication. The ultimate goal is to elucidate the cytopathological basis of lymphoid tissues in PCV2-infected animals based on the cellular responses and the underlying signal pathway(s). The findings thus achieved will help better understand the pathogenesis of PCV2 infections and provide clues to targets for screening novel antiviral chemicals.

猪圆环病毒2型（PCV2）是导致断奶仔猪多系统衰竭综合症的病原，给养猪业造成严重经济损失。PCV2可引起细胞凋亡，我们发现PCV2也可诱导细胞自噬，并以此促进其自身复制。但PCV2诱导上述细胞反应以及PCV2感染淋巴组织的病理学机制还有待深入研究。本项目拟系统研究：（1）PCV2感染和表达病毒蛋白的真核质粒转染细胞的内质网应激、自噬和凋亡及其介导的信号传导途径；（2）PCV2编码蛋白引起相关细胞反应的主要结构域；（3）构建该结构域突变的重组PCV2，感染体外培养细胞和小鼠，解析PCV2感染引起宿主细胞自噬或/和凋亡以及淋巴样组织的病理学变化的机制;（4）比较分析组织细胞中病毒复制与相关细胞反应的关系。通过解析PCV2介导细胞反应的特征和信号传导途径，解明PCV2感染引起宿主组织病变的细胞病理学基础。研究结果为深入研究PCV2的致病机制、探寻抗PCV2药物筛选的靶标奠定基础。

猪圆环病毒2型（PCV2）是导致断奶仔猪多系统衰竭综合症的病原，给养猪业造成严重经济损失。PCV2可引起细胞凋亡，我们发现PCV2也可诱导细胞自噬，并以此促进其自身复制。但PCV2诱导上述细胞反应以及PCV2感染淋巴组织的病理学机制还有待深入研究。本研究（1）阐明了PCV2感染能够诱导非折叠蛋白反应（UPR），并通过PERK/eIF2α/ATF4/CHOP/Bcl-2通路和钙离子外流诱导细胞凋亡，且Cap是PCV2诱导细胞UPR和凋亡的功能蛋白；（2）解明了PCV2能利用PERK通路和GRP78增强其复制，说明PCV2感染早期通过调节UPR利于其在细胞内的增殖；（3）阐明了PCV2感染诱导PK-15细胞自噬的分子机制，即PCV2感染经由CaMKKβ激活AMPK及其下游通路诱导PK-15细胞自噬，且PCV2通过Cap蛋白经由IP3R-Ca2+途径激活CaMKKβ诱导自噬；（4）发现PCV2感染也可以激活CaMKKβ/CaMKI/WIPI1自噬通路，且不依赖于AMPK；（5）以BALB/c小鼠为模型研究解析PCV2感染小鼠能否诱导自噬，及其对PCV2自身复制的影响。PCV2感染小鼠诱导细胞自噬可能CaMKKβ-WIPI1途径，而并未激活AMPK-mTOR自噬通路。主要结论：1）PCV2感染早期通PERK/eIF2α通路诱导UPR，继而通过ATF4/CHOP/Bcl-2通路介导细胞凋亡；2）PCV2感染诱导细胞内钙离子增加，激活IP3R-Ca2+途径和CaMKKβ-AMPK 和 CaMKKβ/CaMKI/WIPI1两条通路诱导自噬；3）PCV2感染也可能通过细胞内钙离子紊乱，激活IP3R途径和Caspase酶系统诱导凋亡。研究结果为诠释PCV2的致病机理及抗PCV2药物的研发提供了基础。