登革病毒 (DENV) 在分類上屬於黃病毒科 (Flaviviridae) 中的黃病毒屬 (Flavivirus)。當宿主遭受登革病毒感染時，被感染的細胞會將登革病毒的非結構蛋1 (NS1) 釋放到細胞外，且此現象與登革重症息息相關。然而目前針對細胞釋放DENV NS1的機制卻尚未研究透徹。在本研究中我們建立了兩株可以穩定表現DENV NS1 的A549以及L929細胞株，用以研究細胞分泌DENV NS1的相關機制。我們先利用酵素結合免疫吸附分析法 (ELISA)，確定這兩株細胞能分泌可溶性的DENV NS1。研究指出，細胞自噬所媒介的非傳統分泌路徑會參與細胞釋放胞內物質。根據實驗結果，我們發現當給予細胞自噬的抑制劑，例如3-Methyladenine (3MA) 以及Bafilomycin A1 (BAF-A1) 時，DENV NS1的分泌會有顯著的下降；而此現象也可以在缺乏與細胞自噬相關的蛋白質，例如Beclin-1以及LC3，的細胞中被觀察到。相反地，當增強細胞自噬作用時，DENV NS1的分泌也會隨之增加。透過免疫染色法，我們也發現DENV NS1會在內質網-粒線體交界處與Atg14以及Beclin-1有交互作用。作為分泌型細胞自噬的運送蛋白質，我們可以在表現NS1的A549細胞的自噬小體中發現DENV NS1的存在。此外，在表現NS1的A549、L929細胞以及被DENV感染的A549細胞中， DENV NS1與具調控細胞自噬作用的G蛋白，Rab37，出現共定位的現象。此外，我們也發現在缺乏Rab37的細胞中，DENV NS1的分泌量也會有顯著性的下降。統合以上初步的結果，我們認為細胞自噬作用與Rab37在協助DENV NS1的分泌中可能扮演了重要的角色。這些發現對於未來在治療登革熱的療程上提供了新的治療標的。

Dengue virus (DENV) is a Flavivirus belonging to Flaviviridae. Upon DENV infection, the non-structural protein 1 (NS1) of DENV will be secreted from DENV-infected cells, which is associated with the pathogenesis of severe dengue. However, the pathway of how DENV NS1 secreted from host cells is not fully understood. We investigated the secretory pathway of DENV NS1 in NS1-expressing A549 and L929 cells. The soluble DENV NS1 is detected from both pNS1-A549 and pNS1-L929 cells by ELISA. It has been reported that an autophagy-associated unconventional pathway is involved in the secretion of cellular protein. According to our results, the secretion of DENV NS1 from pNS1-A549 cells is significantly decreased by treating with autophagy inhibitors, 3-methyladenine, and bafilomycin A1, as well as by silencing autophagy-related proteins, beclin-1 and LC3, while increased secreted NS1 is found by enhancing autophagy activity. By immunostaining assays, DENV NS1 is found not only to interact with Atg14L in the endoplasmic reticulum-mitochondria contact sites but also beclin-1. As a cargo protein of secretory autophagy, DENV NS1 is found in isolated autophagosomes of NS1-expressing cells. Furthermore, an autophagy-regulatory small GTPase, Rab37, is found to colocalize with DENV NS1 in pNS1-A549, pNS1-L929, and DENV-infected cells. Furthermore, the secretion of DENV NS1 is decreased in Rab37-deficient cells. In conclusion, these preliminary results show the potential roles of autophagy and Rab37 in promoting DENV NS1 secretion. These findings may provide the therapeutic targets for treating dengue.

1. Organization, W.H., et al., Dengue: guidelines for diagnosis, treatment, prevention and control. 2009: World Health Organization.
2. Diamond, M.S. and T.C. Pierson, Molecular insight into dengue virus pathogenesis and its implications for disease control. Cell, 2015. 162(3): p. 488-492.
3. Sinkins, S.P. and Gould F, Gene drive systems for insect disease vectors. Nat Rev Genet., 2006. 7(6): p. 427.
4. Lee, K., et al., Clinical characteristics, risk factors, and outcomes in adults experiencing dengue hemorrhagic fever complicated with acute renal failure. Am J Trop Med Hyg., 2009. 80(4): p. 651-655.
5. Guzman, M.G. and S. Vazquez, The complexity of antibody-dependent enhancement of dengue virus infection. Viruses, 2010. 2(12): p. 2649-2662.
6. Guzman, M.G., et al., Dengue: a continuing global threat. Nat Rev Microbiol., 2010. 8(12supp): p. S7.
7. Henchal, E.A. and J.R. Putnak, The dengue viruses. Clin Microbiol Rev., 1990. 3(4): p. 376-396.
8. Itakura, E., C. Kishi-Itakura, and N. Mizushima, The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell, 2012. 151(6): p. 1256-1269.
9. Nemésio, H. and J. Villalaín, Membranotropic regions of the dengue virus prM protein. Biochemistry, 2014. 53(32): p. 5280-5289.
10. Lai, C.-Y., et al., Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J Virol., 2008. 82(13): p. 6631-6643.
11. Kudlacek, S.T. and S.W. Metz, Focused dengue vaccine development: outwitting nature's design. Pathog Dis., 2019. 77(1): p. ftz003.
12. Idrees, S. and U.A. Ashfaq, A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan. Genet Vaccines Ther., 2012. 10(1): p. 6.
13. Xu, H., et al., Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1: implications for early diagnosis and serotyping of dengue virus infections. J Clin Microbiol., 2006. 44(8): p. 2872-2878.
14. Chuang, Y.-C., et al., Re-evaluation of the pathogenic roles of nonstructural protein 1 and its antibodies during dengue virus infection. J Biomed Sci., 2013. 20(1): p. 42.
15. Muñoz-Jordán, J.L., et al., Inhibition of interferon signaling by dengue virus. Proc Natl Acad Sci U S A., 2003. 100(24): p. 14333-14338.
16. Falgout, B., et al., Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol., 1991. 65(5): p. 2467-2475.
17. Benarroch, D., et al., The RNA helicase, nucleotide 5′-triphosphatase, and RNA 5′-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core. Virology, 2004. 328(2): p. 208-218.
18. Stern, O., et al., An N-terminal amphipathic helix in dengue virus nonstructural protein 4A mediates oligomerization and is essential for replication. J Virol., 2013. 87(7): p. 4080-4085.
19. Wan, S.-W., et al., Autoimmunity in dengue pathogenesis. J Formos Med Assoc., 2013. 112(1): p. 3-11.
20. Ackermann, M. and R. Padmanabhan, De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase. J Biol Chem., 2001. 276(43): p. 39926-39937.
21. Su, C.-I., et al., SUMO modification stabilizes dengue virus nonstructural protein 5 to support virus replication. J Virol., 2016. 90(9): p. 4308-4319.
22. Morrison, J. and A. García-Sastre, STAT2 signaling and dengue virus infection. JAKSTAT., 2014. 3(1): p. e27715.
23. Lee, Y.-R., et al., Dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of IL-6 and RANTES. Virus Res., 2007. 126(1-2): p. 216-225.
24. Lin, C.-F., et al., Expression of cytokine, chemokine, and adhesion molecules during endothelial cell activation induced by antibodies against dengue virus nonstructural protein 1. J Immunol., 2005. 174(1): p. 395-403.
25. Tassaneetrithep, B., et al., DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med., 2003. 197(7): p. 823-829.
26. Meertens, L., et al., The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry. Cell Host Microbe., 2012. 12(4): p. 544-557.
27. Reyes-del Valle, J., et al., Dengue virus cellular receptors and tropism. Current Tropical Medicine Reports, 2014. 1(1): p. 36-43.
28. Muller, D.A. and P.R. Young, The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker. Antiviral Res., 2013. 98(2): p. 192-208.
29. Rodenhuis-Zybert, I.A., J. Wilschut, and J.M. Smit, Dengue virus life cycle: viral and host factors modulating infectivity. Cell Mol Life Sci., 2010. 67(16): p. 2773-2786.
30. Screaton, G., et al., New insights into the immunopathology and control of dengue virus infection. Nat Rev Immunol., 2015. 15(12): p. 745.
31. Martina, B.E., P. Koraka, and A.D. Osterhaus, Dengue virus pathogenesis: an integrated view. Clin Microbiol Rev. 2009. 22(4): p. 564-581.
32. Godói, I.P., et al., CYD-TDV dengue vaccine: systematic review and meta-analysis of efficacy, immunogenicity and safety. J Comp Eff Res., 2017. 6(2): p. 165-180.
33. Halstead, S.B. and M. Aguiar, Dengue vaccines: Are they safe for travelers? Travel Med Infect Dis., 2016. 14(4): p. 378-383.
34. Chuang, Y.-C., et al., Dengue virus nonstructural protein 1–induced antibodies cross-react with human plasminogen and enhance its activation. J Immunol., 2016. 196(3): p. 1218-1226.
35. Falconar, A.K., Antibody responses are generated to immunodominant ELK/KLE-type motifs on the nonstructural-1 glycoprotein during live dengue virus infections in mice and humans: implications for diagnosis, pathogenesis, and vaccine design. Clin. Vaccine Immunol., 2007. 14(5): p. 493-504.
36. Rathakrishnan, A., et al., Cytokine expression profile of dengue patients at different phases of illness. PloS One., 2012. 7(12): p. e52215.
37. Avirutnan, P., et al., Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement. J Infect Dis., 2006. 193(8): p. 1078-1088.
38. Chen, H.R., Y.C. Lai, and T.M. Yeh, Dengue virus non-structural protein 1: a pathogenic factor, therapeutic target, and vaccine candidate. J Biomed Sci., 2018. 25(1): p. 58.
39. Chen, H.R., et al., Dengue Virus Nonstructural Protein 1 Induces Vascular Leakage through Macrophage Migration Inhibitory Factor and Autophagy. PLoS Negl Trop Dis., 2016. 10(7): p. e0004828.
40. Amorim, J.H., et al., The dengue virus non-structural 1 protein: risks and benefits. Virus Res., 2014. 181: p. 53-60.
41. Glasner, D.R., et al., The good, the bad, and the shocking: the multiple roles of dengue virus nonstructural protein 1 in protection and pathogenesis. Annu Rev Virol., 2018. 5: p. 227-253.
42. Modhiran, N., et al., Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity. Sci Transl Med. 2015. 7(304): p. 304ra142.
43. Chen, H.-R., et al., Macrophage migration inhibitory factor is critical for dengue NS1-induced endothelial glycocalyx degradation and hyperpermeability. PLoS Pathog., 2018. 14(4): p. e1007033.
44. Avirutnan, P., et al., Antagonism of the complement component C4 by flavivirus nonstructural protein NS1. J Exp Med., 2010. 207(4): p. 793-806.
45. Avirutnan, P., et al., Binding of flavivirus nonstructural protein NS1 to C4b binding protein modulates complement activation. J Immunol., 2011. 187(1): p. 424-433.
46. Thiemmeca, S., et al., Secreted NS1 protects dengue virus from mannose-binding lectin–mediated neutralization. J Immunol., 2016. 197(10): p. 4053-4065.
47. Flamand, M., et al., Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. J Virol., 1999. 73(7): p. 6104-6110.
48. Alcala, A.C., et al., The dengue virus non-structural protein 1 (NS1) is secreted efficiently from infected mosquito cells. Virology, 2016. 488: p. 278-87.
49. Alcala, A.C., et al., The dengue virus non-structural protein 1 (NS1) is secreted from infected mosquito cells via a non-classical caveolin-1-dependent pathway. J Gen Virol., 2017. 98(8): p. 2088-2099.
50. Kim, J., H.Y. Gee, and M.G. Lee, Unconventional protein secretion–new insights into the pathogenesis and therapeutic targets of human diseases. J Cell Sci., 2018. 131(12): p. jcs213686.
51. Rabouille, C., V. Malhotra, and W. Nickel, Diversity in unconventional protein secretion. 2012, The Company of Biologists Ltd.
52. Claude-Taupin, A., et al., Autophagy’s secret life: secretion instead of degradation. Essays Biochem., 2017. 61(6): p. 637-647.
53. Zhang, M. and R. Schekman, Unconventional secretion, unconventional solutions. Science., 2013. 340(6132): p. 559-561.
54. Zhang, M., et al., Translocation of interleukin-1β into a vesicle intermediate in autophagy-mediated secretion. Elife, 2015. 4: p. e11205.
55. Dupont, N., et al., Autophagy‐based unconventional secretory pathway for extracellular delivery of IL‐1β. EMBO J., 2011. 30(23): p. 4701-4711.
56. Chen, Y.-D., et al., Exophagy of annexin A2 via RAB11, RAB8A and RAB27A in IFN-γ-stimulated lung epithelial cells. Sci Rep., 2017. 7(1): p. 5676.
57. Nilsson, P., et al., Aβ secretion and plaque formation depend on autophagy. Cell Rep., 2013. 5(1): p. 61-69.
58. Son, S.M., et al., Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease. Autophagy, 2016. 12(5): p. 784-800.
59. Bel, S., et al., Paneth cells secrete lysozyme via secretory autophagy during bacterial infection of the intestine. Science, 2017. 357(6355): p. 1047-1052.
60. Bird, S.W., et al., Nonlytic viral spread enhanced by autophagy components. Proc Natl Acad Sci U S A., 2014. 111(36): p. 13081-13086.
61. Robinson, S.M., et al., Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers. PLoS Pathog., 2014. 10(4): p. e1004045.
62. Jackson, W.T., et al., Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol, 2005. 3(5): p. e156.
63. Chen, Y.-H., et al., Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses. Cell, 2015. 160(4): p. 619-630.
64. Shrivastava, S., et al., Knockdown of autophagy inhibits infectious hepatitis C virus release by the exosomal pathway. J Virol., 2016. 90(3): p. 1387-1396.
65. Yu, L., Y. Chen, and S.A. Tooze, Autophagy pathway: cellular and molecular mechanisms. Autophagy, 2018. 14(2): p. 207-215.
66. Lamb, C.A., T. Yoshimori, and S.A. Tooze, The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol., 2013. 14(12): p. 759-74.
67. Rubinsztein, D.C., T. Shpilka, and Z. Elazar, Mechanisms of autophagosome biogenesis. Curr Biol., 2012. 22(1): p. R29-R34.
68. Shibutani, S.T. and T. Yoshimori, A current perspective of autophagosome biogenesis. Cell Res., 2014. 24(1): p. 58.
69. Ponpuak, M., et al., Secretory autophagy. Curr Opin Cell Biol., 2015. 35: p. 106-116.
70. Heaton, N.S., G. Randall, Dengue virus-induced autophagy regulates lipid metabolism. Cell Host Microbe., 2010. 8(5): p. 422-432.
71. Heaton, N.S. and G. Randall, Dengue virus and autophagy. Viruses., 2011. 3(8): p. 1332-1341.
72. Wu, Y.W., et al., Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization. Sci Rep., 2016. 6: p. 32243.
73. McLean, J.E., et al., Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication. J Biol Chem., 2011. 286(25): p. 22147-59.
74. Lee, Y.-R., et al., Autophagic machinery activated by dengue virus enhances virus replication. Virology., 2008. 374(2): p. 240-248.
75. Stenmark, H., Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol., 2009. 10: p. 513.
76. Krishnan, M.N., et al., Rab 5 is required for the cellular entry of dengue and West Nile viruses. J Virol., 2007. 81(9): p. 4881-4885.
77. Xu, X.-F., et al., Rab8, a vesicular traffic regulator, is involved in dengue virus infection in HepG2 cells. Intervirology, 2008. 51(3): p. 182-188.
78. Tang, W.-C., et al., Rab18 facilitates dengue virus infection by targeting fatty acid synthase to sites of viral replication. J Virol., 2014. 88(12): p. 6793-6804.
79. Mori, R., et al., Release of TNF‐α from macrophages is mediated by small GTPase Rab37. Eur J Immunol., 2011. 41(11): p. 3230-3239.
80. Ljubicic, S., et al., The GTPase Rab37 participates in the control of insulin exocytosis. PLoS One., 2013. 8(6): p. e68255.
81. Masuda, E.S., et al., Rab37 is a novel mast cell specific GTPase localized to secretory granules. FEBS Lett., 2000. 470(1): p. 61-64.
82. Higashio, H., Y.-i. Satoh, and T. Saino, Mast cell degranulation is negatively regulated by the Munc13-4-binding small-guanosine triphosphatase Rab37. Sci Rep., 2016. 6: p. 22539.
83. Tsai, C.-H., et al., Small GTPase Rab37 targets tissue inhibitor of metalloproteinase 1 for exocytosis and thus suppresses tumour metastasis. Nat Commun., 2014. 5: p. 4804.
84. Tzeng, H.-T., et al., Dysregulation of Rab37-mediated cross-talk between cancer cells and endothelial cells via thrombospondin-1 promotes tumor neovasculature and metastasis. Clin Cancer Res., 2017. 23(9): p. 2335-2345.
85. Cho, S.-H., et al., Rab37 mediates exocytosis of secreted frizzled-related protein 1 to inhibit Wnt signaling and thus suppress lung cancer stemness. Cell Death Dis., 2018. 9(9): p. 868.
86. Tzeng, H.T., et al., Rab37 in lung cancer mediates exocytosis of soluble ST2 and thus skews macrophages toward tumor‐suppressing phenotype. Int J Cancer., 2018. 143(7): p. 1753-1763.
87. Sheng, Y., et al., RAB37 interacts directly with ATG5 and promotes autophagosome formation via regulating ATG5-12-16 complex assembly. Cell Death Differ., 2018 .25(5):918-934
88. Song, Y., et al., The small GTPase RAB37 functions as an organizer for autophagosome biogenesis. Autophagy., 2018. 14(4): p. 727-729.
89. Kabeya, Y., et al., LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J., 2000. 19(21): p. 5720-5728.
90. Lee, Y.-R., et al., Dengue virus infection induces autophagy: an in vivo study. J Biomed Sci., 2013. 20(1): p. 65.
91. Hamasaki, M., et al., Autophagosomes form at ER–mitochondria contact sites. Nature, 2013. 495(7441): p. 389.
92. Matsunaga, K., et al., Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L. J Cell Biol., 2010. 190(4): p. 511-521.
93. Santiago-Tirado, F.H. and A. Bretscher, Membrane-trafficking sorting hubs: cooperation between PI4P and small GTPases at the trans-Golgi network. Trends Cell Biol., 2011. 21(9): p. 515-525.
94. Crooks, A.J., et al., The NS1 protein of tick-borne encephalitis virus forms multimeric species upon secretion from the host cell. J Gen Virol., 1994. 75(12): p. 3453-3460.
95. Nishida, Y., et al., Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature, 2009. 461(7264): p. 654.
96. Winkler, G., et al., Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology, 1989. 171(1): p. 302-305.
97. Watterson, D., N. Modhiran, and P.R. Young, The many faces of the flavivirus NS1 protein offer a multitude of options for inhibitor design. Antiviral Res., 2016. 130: p. 7-18.
98. Yap, S.S., et al., Dengue virus glycosylation: what do we know? Front Microbiol., 2017. 8: p. 1415.
99. Gutsche, I., et al., Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proc Natl Acad Sci U S A., 2011. 108(19): p. 8003-8008.
100. Mason, P.W., Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells. Virology, 1989. 169(2): p. 354-364.
101. Panyasrivanit, M., et al., Linking dengue virus entry and translation/replication through amphisomes. Autophagy, 2009. 5(3): p. 434-435.
102. Panyasrivanit, M., et al., Co-localization of constituents of the dengue virus translation and replication machinery with amphisomes. J Gen Virol., 2009. 90(2): p. 448-456.
103. Johansen, T. and T. Lamark, Selective autophagy mediated by autophagic adapter proteins. Autophagy, 2011. 7(3): p. 279-296.
104. Mauvezin, C. and T.P. Neufeld, Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion. Autophagy, 2015. 11(8): p. 1437-1438.
105. Weisz, O.A., Acidification and protein traffic. International review of cytology, 2003: p. 259-320.
106. Manna, D., et al., Endocytic Rab proteins are required for hepatitis C virus replication complex formation. Virology., 2010. 398(1): p. 21-37.
107. Spearman, P.J.S.G., Viral interactions with host cell Rab GTPases. Small GTPases., 2018. 9(1-2): p. 192-201.
108. Sheng, Y., et al., RAB37 interacts directly with ATG5 and promotes autophagosome formation via regulating ATG5-12-16 complex assembly. Cell Death Differ., 2018. 25(5): p. 918-934.