腸病毒A71型(EV-A71)感染可引起嚴重的神經系統併發症並導致幼兒死亡。EV-A71可誘導細胞自噬增加病毒複製。細胞自噬(autophagy)包含三個主要的步驟：自噬體形成(autophagosome formation)、自噬體與溶酶體融合(autophagosome-lysosome fusion)及溶酶體降解。我們近期的體外研究發現透過syntaxin 17基因剔除去抑制自噬體與溶酶體/內胞體(endosome)融合或leupeptin處理細胞去抑制溶酶體降解，減少EV-A71基因組複製與病毒產量。在本篇研究中，我們進一步發現syntaxin 17基因剔除及leupeptin處理細胞皆會減少EV-A71脫殼(uncoating)，但不影響病毒貼附與進入細胞的步驟。此外，利用抑制囊泡酸化及細胞自噬的抑制劑bafilomycin A1，也能減少病毒的脫殼及產量。因此，應用基因剔除或細胞自噬抑制劑去抑制細胞內的溶酶體活性會導致EV-A71脫殼及複製減少。為了揭示溶酶體增強病毒感染的機制，我們找出負責EV-A71脫殼的蛋白酶並研究它們的重要性。組織蛋白酶B (cathepsin B)或組織蛋白酶L (cathepsin L)已被發現會促進其他病毒的脫殼且其功能可被leupeptin抑制。使用針對cathepsin B或cathepsin L的抑制劑可減少EV-A71脫殼及產量。我們也顯示cathepsin B或cathepsin L可以切割EV-A71外殼蛋白VP1及VP2。總體而言，我們的研究強調溶酶體蛋白酶對於EV-A71脫殼的重要性以及溶酶體在細胞自噬中如何透過促進脫殼來增強EV-A71感染。

Enterovirus A71 (EV-A71) infection can cause severe neurological complication and lead to the death of young children. EV-A71 can induce autophagy to increase viral replication. Three major steps, including formation of autophagosome, autophagosome-lysosome fusion, and lysosomal degradation are involved in autophagy. Our previous in vitro studies found that inhibition of autophagosome-lysosome/endosome fusion or lysosomal degradation by syntaxin 17 knockdown or leupeptin treatment reduced EV-A71 genome replication and yields. In the present study, we found that syntaxin 17 knockdown and leupeptin treatment reduced EV-A71 uncoating, but failed to affect virus binding and entry into cells. In addition, using bafilomycin A1, a vesicle acidification and autophagy inhibitor, also reduced virus uncoating and yields. Accordingly, application of genetic approaches or autophagy inhibitors to suppress lysosomal activities in cells leads to the reduction of EV-A71 replication with a decrease of virus uncoating. To uncover the mechanism by which lysosome functions to enhance virus infection, we identified lysosomal proteases responsible for EV-A71 uncoating and investigated the significance. Treatment of cells with inhibitors against cathepsin B or cathepsin L, which promote uncoating of other viruses and are inhibited by leupeptin, reduced EV-A71 uncoating and yields. We also showed that cathepsin B or cathepsin L can cleave EV-A71 outer capsid proteins, VP1 and VP2. Overall, our study highlight the importance of lysosome proteases in EV-A71 uncoating and how lysosome functions in autophagy to enhance EV-A71 replication by promoting uncoating.

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