腸病毒71型(EV71)感染會造成幼童產生嚴重的神經系統併發症甚至死亡，特別是在亞太地區，包括台灣。目前仍缺乏針對EV71的抗病毒治療和疫苗，因此，研究EV71的致病機制是迫切需要的。細胞自噬(autophagy)分解回收細胞質的大分子來維持細胞的恆定。完整的細胞自噬包含三個主要的階段: 自噬體(autophagosome)的形成、自噬體和溶小體(lysosome)融合、以及溶小體降解。許多正股RNA病毒，包括EV71，可以利用細胞自噬來幫助病毒複製。先前的研究顯示EV71的感染會誘發細胞自噬流(autophagic flux)，然而，細胞自噬的每個階段對於病毒RNA複製、蛋白合成、病毒顆粒的生產和釋放的重要性尚未被發表。在本篇研究中，我們確認了EV71會誘發人類細胞株產生細胞自噬流。為了證實細胞自噬對病毒複製是否重要，我們藉由抑制自噬體(autophagosome)的形成、自噬體和溶小體(lysosome)融合、以及溶小體降解，揭示了細胞自噬如何幫助EV71複製，並且提供策略制定有效的抗病毒治療。

Enterovirus 71 (EV71) infection can cause deaths and severe neurological complications in young children, especially in the Asia-Pacific region including Taiwan. Currently, the lack of antiviral therapies and vaccines specific for EV71 stresses the need to study viral pathogenesis. Autophagy maintains homeostasis by recycling cytoplasmic materials. The complete autophagic flux contains three major steps: autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation. Several positive-stranded RNA viruses, including EV71, can take advantage of autophagy for their own benefits. Previous studies showed that EV71 infection induces complete autophagic flux. However, the importance of each autophagic step in EV71 RNA replication, protein synthesis, virus production, and egress has not been addressed. In the present study, we confirmed that EV71 infection induces autophagic flux in human cell lines. To elucidate whether autophagy is essential to EV71 replication, we inhibited three major autophagic steps: autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation. Our findings uncover how autophagy contributes to EV71 replication, and provide strategies for developing effective antiviral therapies.

REFERENCES
1. Shih SR, Stollar V, Li ML. 2011. Host factors in enterovirus 71 replication. J Virol 85:9658-9666.
2. Yamayoshi S, Yamashita Y, Li J, Hanagata N, Minowa T, Takemura T, Koike S. 2009. Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat Med 15:798-801.
3. Nishimura Y, Shimojima M, Tano Y, Miyamura T, Wakita T, Shimizu H. 2009. Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat Med 15:794-797.
4. Lin TY, Chang LY, Hsia SH, Huang YC, Chiu CH, Hsueh C, Shih SR, Liu CC, Wu MH. 2002. The 1998 enterovirus 71 outbreak in Taiwan: pathogenesis and management. Clin Infect Dis 34 Suppl 2:S52-57.
5. Ooi MH, Wong SC, Lewthwaite P, Cardosa MJ, Solomon T. 2010. Clinical features, diagnosis, and management of enterovirus 71. Lancet Neurol 9:1097-1105.
6. Chang LY, Huang LM, Gau SS, Wu YY, Hsia SH, Fan TY, Lin KL, Huang YC, Lu CY, Lin TY. 2007. Neurodevelopment and cognition in children after enterovirus 71 infection. N Engl J Med 356:1226-1234.
7. Gau SS, Chang LY, Huang LM, Fan TY, Wu YY, Lin TY. 2008. Attention-deficit/hyperactivity-related symptoms among children with enterovirus 71 infection of the central nervous system. Pediatrics 122:e452-458.
8. Huang MC, Wang SM, Hsu YW, Lin HC, Chi CY, Liu CC. 2006. Long-term cognitive and motor deficits after enterovirus 71 brainstem encephalitis in children. Pediatrics 118:e1785-1788.
9. Li J, Chen F, Liu T, Wang L. 2012. MRI findings of neurological complications in hand-foot-mouth disease by enterovirus 71 infection. Int J Neurosci 122:338-344.
10. Zhang YC, Li XW, Zhu XD, Qian SY, Shang YX, Li BR, Liu XL. 2010. Clinical characteristics and treatment of severe encephalitis associated with neurogenic pulmonary edema caused by enterovirus 71 in China. World J Emerg Med 1:108-113.
11. Lee MS, Lin TY, Chiang PS, Li WC, Luo ST, Tsao KC, Liou GY, Huang ML, Hsia SH, Huang YC, Chang SC. 2010. An investigation of epidemic enterovirus 71 infection in Taiwan, 2008: clinical, virologic, and serologic features. Pediatr Infect Dis J 29:1030-1034.
12. Zhu FC, Meng FY, Li JX, Li XL, Mao QY, Tao H, Zhang YT, Yao X, Chu K, Chen QH, Hu YM, Wu X, Liu P, Zhu LY, Gao F, Jin H, Chen YJ, Dong YY, Liang YC, Shi NM, Ge HM, Liu L, Chen SG, Ai X, Zhang ZY, Ji YG, Luo FJ, Chen XQ, Zhang Y, Zhu LW, Liang ZL, Shen XL. 2013. Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 381:2024-2032.
13. Zhu F, Xu W, Xia J, Liang Z, Liu Y, Zhang X, Tan X, Wang L, Mao Q, Wu J, Hu Y, Ji T, Song L, Liang Q, Zhang B, Gao Q, Li J, Wang S, Hu Y, Gu S, Zhang J, Yao G, Gu J, Wang X, Zhou Y, Chen C, Zhang M, Cao M, Wang J, Wang H, Wang N. 2014. Efficacy, safety, and immunogenicity of an enterovirus 71 vaccine in China. N Engl J Med 370:818-828.
14. Li R, Liu L, Mo Z, Wang X, Xia J, Liang Z, Zhang Y, Li Y, Mao Q, Wang J, Jiang L, Dong C, Che Y, Huang T, Jiang Z, Xie Z, Wang L, Liao Y, Liang Y, Nong Y, Liu J, Zhao H, Na R, Guo L, Pu J, Yang E, Sun L, Cui P, Shi H, Wang J, Li Q. 2014. An inactivated enterovirus 71 vaccine in healthy children. N Engl J Med 370:829-837.
15. Yang Z, Klionsky DJ. 2010. Eaten alive: a history of macroautophagy. Nat Cell Biol 12:814-822.
16. Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo S, Massey DC, Menzies FM, Moreau K, Narayanan U, Renna M, Siddiqi FH, Underwood BR, Winslow AR, Rubinsztein DC. 2010. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90:1383-1435.
17. Mizushima N, Komatsu M. 2011. Autophagy: renovation of cells and tissues. Cell 147:728-741.
18. Wileman T. 2013. Autophagy as a defence against intracellular pathogens. Essays Biochem 55:153-163.
19. Glick D, Barth S, Macleod KF. 2010. Autophagy: cellular and molecular mechanisms. The Journal of Pathology 221:3-12.
20. Kraft C, Martens S. 2012. Mechanisms and regulation of autophagosome formation. Curr Opin Cell Biol 24:496-501.
21. Mizushima N, Yoshimori T, Ohsumi Y. 2011. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107-132.
22. Walczak M, Martens S. 2013. Dissecting the role of the Atg12-Atg5-Atg16 complex during autophagosome formation. Autophagy 9:424-425.
23. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T. 2000. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720-5728.
24. Itakura E, Kishi-Itakura C, Mizushima N. 2012. The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151:1256-1269.
25. Jiang P, Nishimura T, Sakamaki Y, Itakura E, Hatta T, Natsume T, Mizushima N. 2014. The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17. Mol Biol Cell 25:1327-1337.
26. Fasshauer D, Sutton RB, Brunger AT, Jahn R. 1998. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc Natl Acad Sci U S A 95:15781-15786.
27. Mizushima N. 2007. Autophagy: process and function. Genes Dev 21:2861-2873.
28. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, Agholme L, Agnello M, Agostinis P, Aguirre-Ghiso JA, Ahn HJ, Ait-Mohamed O, Ait-Si-Ali S, Akematsu T, Akira S, Al-Younes HM, Al-Zeer MA, Albert ML, Albin RL, Alegre-Abarrategui J, Aleo MF, Alirezaei M, Almasan A, Almonte-Becerril M, Amano A, Amaravadi R, Amarnath S, Amer AO, Andrieu-Abadie N, Anantharam V, Ann DK, Anoopkumar-Dukie S, Aoki H, Apostolova N, Arancia G, Aris JP, Asanuma K, Asare NY, Ashida H, Askanas V, Askew DS, Auberger P, Baba M, Backues SK, Baehrecke EH, Bahr BA, Bai XY, Bailly Y, Baiocchi R, Baldini G, et al. 2012. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445-544.
29. Tanida I. 2011. Autophagosome formation and molecular mechanism of autophagy. Antioxid Redox Signal 14:2201-2214.
30. den Boon JA, Ahlquist P. 2010. Organelle-like membrane compartmentalization of positive-strand RNA virus replication factories. Annu Rev Microbiol 64:241-256.
31. Miller S, Krijnse-Locker J. 2008. Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol 6:363-374.
32. Jackson WT, Giddings TH, Jr., Taylor MP, Mulinyawe S, Rabinovitch M, Kopito RR, Kirkegaard K. 2005. Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol 3:e156.
33. Wileman T. 2006. Aggresomes and autophagy generate sites for virus replication. Science 312:875-878.
34. Denison MR. 2008. Seeking membranes: positive-strand RNA virus replication complexes. PLoS Biol 6:e270.
35. Wong J, Zhang J, Si X, Gao G, Mao I, McManus BM, Luo H. 2008. Autophagosome supports coxsackievirus B3 replication in host cells. J Virol 82:9143-9153.
36. Richards AL, Jackson WT. 2012. Intracellular vesicle acidification promotes maturation of infectious poliovirus particles. PLoS Pathog 8:e1003046.
37. Khakpoor A, Panyasrivanit M, Wikan N, Smith DR. 2009. A role for autophagolysosomes in dengue virus 3 production in HepG2 cells. J Gen Virol 90:1093-1103.
38. Ke PY, Chen SS. 2011. Activation of the unfolded protein response and autophagy after hepatitis C virus infection suppresses innate antiviral immunity in vitro. J Clin Invest 121:37-56.
39. Taguwa S, Kambara H, Fujita N, Noda T, Yoshimori T, Koike K, Moriishi K, Matsuura Y. 2011. Dysfunction of autophagy participates in vacuole formation and cell death in cells replicating hepatitis C virus. J Virol 85:13185-13194.
40. Kemball CC, Alirezaei M, Flynn CT, Wood MR, Harkins S, Kiosses WB, Whitton JL. 2010. Coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo. J Virol 84:12110-12124.
41. Dang M, Wang X, Wang Q, Wang Y, Lin J, Sun Y, Li X, Zhang L, Lou Z, Wang J, Rao Z. 2014. Molecular mechanism of SCARB2-mediated attachment and uncoating of EV71. Protein Cell 5:692-703.
42. Lee YR, Wang PS, Wang JR, Liu HS. 2014. Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model. J Biomed Sci 21:80.
43. Huang SC, Chang CL, Wang PS, Tsai Y, Liu HS. 2009. Enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replication. J Med Virol 81:1241-1252.
44. Xi X, Zhang X, Wang B, Wang T, Wang J, Huang H, Wang J, Jin Q, Zhao Z. 2013. The interplays between autophagy and apoptosis induced by enterovirus 71. PLoS One 8:e56966.
45. Li ZH, Li CM, Ling P, Shen FH, Chen SH, Liu CC, Yu CK, Chen SH. 2008. Ribavirin reduces mortality in enterovirus 71-infected mice by decreasing viral replication. J Infect Dis 197:854-857.
46. Yamayoshi S, Ohka S, Fujii K, Koike S. 2013. Functional comparison of SCARB2 and PSGL1 as receptors for enterovirus 71. J Virol 87:3335-3347.
47. Lin YW, Lin HY, Tsou YL, Chitra E, Hsiao KN, Shao HY, Liu CC, Sia C, Chong P, Chow YH. 2012. Human SCARB2-mediated entry and endocytosis of EV71. PLoS One 7:e30507.
48. Luzio JP, Rous BA, Bright NA, Pryor PR, Mullock BM, Piper RC. 2000. Lysosome-endosome fusion and lysosome biogenesis. J Cell Sci 113 ( Pt 9):1515-1524.
49. Ebert DH, Deussing J, Peters C, Dermody TS. 2002. Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells. J Biol Chem 277:24609-24617.
50. Akache B, Grimm D, Shen X, Fuess S, Yant SR, Glazer DS, Park J, Kay MA. 2007. A two-hybrid screen identifies cathepsins B and L as uncoating factors for adeno-associated virus 2 and 8. Mol Ther 15:330-339.
51. Magliano D, Marshall JA, Bowden DS, Vardaxis N, Meanger J, Lee JY. 1998. Rubella virus replication complexes are virus-modified lysosomes. Virology 240:57-63.
52. Schlegel A, Giddings TH, Jr., Ladinsky MS, Kirkegaard K. 1996. Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol 70:6576-6588.
53. Taylor MP, Kirkegaard K. 2007. Modification of cellular autophagy protein LC3 by poliovirus. J Virol 81:12543-12553.
54. Mori Y, Yamashita T, Tanaka Y, Tsuda Y, Abe T, Moriishi K, Matsuura Y. 2007. Processing of capsid protein by cathepsin L plays a crucial role in replication of Japanese encephalitis virus in neural and macrophage cells. J Virol 81:8477-8487.
55. McMinn PC. 2002. An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev 26:91-107.
56. Blok J, Mulder-Stapel AA, Ginsel LA, Daems WT. 1981. The effect of chloroquine on lysosomal function and cell-coat glycoprotein transport in the absorptive cells of cultured human small-intestinal tissue. Cell Tissue Res 218:227-251.
57. Shalini S, Chaudhuri S, Sutton PL, Mishra N, Srivastava N, David JK, Ravindran KJ, Carlton JM, Eapen A. 2014. Chloroquine efficacy studies confirm drug susceptibility of Plasmodium vivax in Chennai, India. Malar J 13:129.
58. Amaratunga C, Sreng S, Mao S, Tullo GS, Anderson JM, Chuor CM, Suon S, Fairhurst RM. 2014. Chloroquine remains effective for treating Plasmodium vivax malaria in Pursat province, Western Cambodia. Antimicrob Agents Chemother 58:6270-6272.