摘要
Porcine circovirus 3(PCV3)has been detected in major pig-producing countries around the world since its first report in the US in 2016.Most current studies have focused on epidemiological investigations and detection methods of PCV3 because of lack of live virus strains for research on its pathogenesis in porcine cells or even in pigs.We constructed a recombinant plasmid pCMV-Cap carrying the PCV3 orf2 gene to investigate the effects of capsid(Cap)protein expression on autophagic response in human embryonic kidney cell line 293 T(HEK293 T).We demonstrate that PCV3 Cap protein induced complete autophagy shown as formation of autophagosomes and autophagosome-like vesicles as well as LC3-II conversion from LC3-I via inhibiting phosphorylation of the mammalian target of rapamycin(mTOR)in HEK293 T cells.The ubiquitin–proteasome pathway is also involved in the autophagy process.These findings provide insight for further exploration of PCV3 pathogenetic mechanisms in porcine cells.
目的:探明猪圆环病毒3型衣壳(Cap)蛋白如何诱导细胞自噬。创新点:首次证明猪圆环病毒3型Cap蛋白诱导细胞自噬,泛素-蛋白酶体途径也参与其中。方法:构建过表达猪圆环病毒3型Cap蛋白的真核表达载体,转染HEK293T细胞,采用免疫印迹检测LC3、p62和m TOR等自噬通路的关键分子。利用激光扫描共聚焦显微镜和透射电镜,观察表达Cap蛋白的细胞是否有EGFP-LC3B点状聚集和自噬泡生成。利用氯喹抑制溶酶体酸化,探究衣壳蛋白是否诱导完整的自噬流。利用MG132抑制蛋白酶体途径,探究泛素-蛋白酶体途径是否参与自噬过程。结论:猪圆环病毒3型Cap蛋白通过抑制雷帕霉素靶蛋白的磷酸化诱导HEK293T细胞自噬,泛素-蛋白酶体途径也参与这一过程。
基金
Project supported by the Zhejiang Provincial Department of Science and Technology(No.2018C02028),China。
