Mechanistic study on Autophagy Induced by Expressions of the Enterovirus 71 2BC Protein

• Main Authors: Yi-Hsuan Chang, 張以暄
• Other Authors: Szu-Hao Kung
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/12900511655314086044

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 104 === Autophagy is a catabolic process involving an intracellular membrane rearrangement that recycles cellular components for cellular homeostasis. EV71 has been reported to subvert autophagy to enhance viral replication both in vitro and in vivo; however, the upstream triggers that initiate the autophagy process have not been elucidated. In this study, we attempted to identify the viral triggers that initiate autophagy, and the signaling pathway that mediates the process. We investigated the viral proteins reported to mediate membrane remodeling in enterovirus-infected cells. Viral 2BC expression induced the highest level of microtubule-associated protein light chain 3 (LC3)-2, a hallmark of autophagosome formation, compared with those induced by viral 2B, 2C and 3A. The result was confirmed by higher number of EGFP-LC3 puncta formed in the 2BC-transfected cells. Our previous study showed that EV71 may induce autophagy through calcium/calmodulin-dependent kinase kinase-β (CaMKK-β)/adenosine monophosphate-activated protein kinase (AMPK) signaling. Therefore, to study mechanism of autophagosome formation triggered by viral 2BC expression, we measured the cytosolic calcium influx by Fluo-8 staining and found that 2BC expression raised the cytosolic calcium concentration. EGFP-LC3 punta formation in the viral 2BC expressing-cells were significantly decreased by calcium chelator, CaMKK-β inhibitor and AMPK inhibitor, supporting that 2BC triggered autophagy flux may mediated by CaMKK-β-AMPK pathway. In addition, we showed that viral 2BC was localized at Golgi apparatus, supporting the role that viral 2BC plays in mediating calcium flow from Golgi apparatus to the cytoplasm. In sum, our data supported that EV71 infection initiates autophagy through viral 2BC protein that localize to Golgi apparatus and elevates cytosolic Ca2+, a scenario that activates the CaMKK-β-AMPK pathway and consequent autophagosome formation.