細胞自噬(autophagy) 是一個維持細胞恆定的過程，透過與溶酶體 (lysosome) 融合以降解並回收老廢物質。先前研究指出，正股RNA病毒可利用細胞自噬幫助病毒複製，登革病毒 (dengue virus, DENV)為其中之一。登革病毒為一具脂質膜 (envelope) 之黃熱病毒 (Flavivirus) 。黃熱病毒在感染細胞後，會在內質網進行複製，由於病毒複製的蛋白質累積進而誘發內質網壓力 (ER stress)．倘若內質網壓力持續發生，細胞會走向凋亡 (apoptosis) 。因此為了緩解內質網壓力，細胞會活化摺疊蛋白反應（unfolded protein response；UPR）並活化其下游之訊息路徑以清除累積的不當摺疊的蛋白，恢復內質網之恆定。實驗室先前之研究顯示，DENV2 感染細胞後會誘發內質網壓力，此一內質網壓力進而依序活化下游三條摺疊蛋白反應訊息路徑，其中，PERK及IRE1更進一步活化自噬反應。若使用IRE1其下游分子JNK磷酸化之抑制劑SP600125，可抑制自噬反應，顯示病毒經由ER stress -> IRE1-> JNK訊息路徑誘發自噬反應並可促進其複製以及加重小鼠之病徵。然而，此一路徑是否影響小鼠的生存率仍不清楚。本研究利用上述之JNK磷酸化抑制劑同樣的可降低DENV2感染小鼠的病徵，並進一步發現可提高其存活率。登革病毒感染之病患會出現骨痛、骨髓細胞減少及血小板低下等臨床表徵。先前文獻則指出，造成此一現象的原因之一是登革病毒感染了骨髓中的血小板前驅細胞 (巨核細胞，megakaryocyte)。已知證據顯示巨核細胞是DENV主要感染的目標細胞，然而感染後造成細胞病變之研究仍付之闕如。本研究探討Meg01巨核細胞株受DENV2感染後之影響。研究發現DENV2會誘發Meg01細胞中之內質網壓力、自噬反應及完整的自噬反應流(autophagy flux)，DENV2誘發Meg01細胞內之自噬反應促使病毒量下降，然而抑制自噬反應並無法增加病毒之複製。我們也觀察了促發炎細胞激素HMGB1在DENV2感染的Meg01細胞中之表現情形，結果顯示DENV2感染後，HMGB1在胞內的表現量隨感染時間而上升，伴隨著分泌到培養液中的HMGB1上升。此外DENV2感染之Meg01細胞體型增大，並呈現多核及染色質異常等現象。總結之，本研究發現DENV會誘發Meg01細胞中之自噬反應、內質網壓力、並影響Meg01細胞的分裂及染色體之穩定性，這些現象可能與登革病毒導致之出血現象有關，值得做更深入之探討。

Dengue virus (DENV) is an envelope virus with single-stranded positive RNA belonging to Flaviviridae. Autophagy is a mechanism to maintain cell homeostasis by degrading components in the autolysosome. Previous studies reported that DENV infection activates autophagy which is beneficial to viral replication. Because Flaviviruses utilize endoplasmic reticulum (ER) for replication, accumulated viral proteins on its surface induces ER stress, and persistent ER stress leads to apoptosis. Therefore, cells trigger the unfolding protein response (UPR) to clean these unfolded proteins. We previously revealed that one of the three UPR pathways IRE-1->JNK->Bcl-2 signaling pathway could induce autophagy. We found that inhibition of JNK pathway by a JNK phosphorylation inhibitor (SP600125) suppressed autophagy, decreased dengue virus replication and alleviated disease symptoms. In this study, we further found that inhibition of IRE-1->JNK->Bcl-2->BECN1-> autophagy signaling pathway by SP600125 not only decreased DENV2 replication, but also alleviated disease symptoms and survival rate in mice. Previous studies reported that DENV infects and propagates in platelet progenitor megakaryocytes. DENV infection in megakaryocytes interferes with their differentiation and maturation, which may lead to clinical thrombopenia in DENV patients. Although megakaryocytes are reported to be the target cells for DENV infection, the effect of DENV infection on megakaryocytes remains unclear. In this study, we revealed that DENV2 infection of Meg01 cells induces ER stress and a complete autophagy flux. Differently, DENV-induced autophagy in Meg01 cells decreased virus replication. Previous studies suggest that secretory autophagy transports high mobility group box 1 (HMGB1) to the extracellular space in the acute inflammation phase. DENV2 infection of Meg01 cells increased the intracellular and the extracellular levels of the proinflammatory cytokine, HMGB1. We further found that in DENV2 infected Meg01 cells, their size was increased, multinuclei and chromosome abnormality. Our findings in DENV2 infected Meg01 cells may be related to the symptoms of thrombopenia and bleeding tendency in severe dengue patients.

1. Nagy PD, Pogany J. 2012. The dependence of viral RNA replication on co-opted host factors. Nature Rev Microbiology 10:137-149.
2. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF, George DB, Jaenisch T, Wint GR, Simmons CP, Scott TW, Farrar JJ, Hay SI. 2013. The global distribution and burden of dengue. Nature 496:504-507.
3. Herrero LJ, Zakhary A, Gahan ME, Nelson MA, Herring BL, Hapel AJ, Keller PA, Obeysekera M, Chen W, Sheng KC, Taylor A, Wolf S, Bettadapura J, Broor S, Dar L, Mahalingam S. 2013. Dengue virus therapeutic intervention strategies based on viral, vector and host factors involved in disease pathogenesis. Pharmacol Ther 137:266-282.
4. Questions and Answers on Dengue Vaccines: Phase III study of CYD-TDENV. 2014 WHO
5. Brian JM, David VE, Ichiro K, Micheal WF, Francis AE. 1991. Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc gamma receptors. J Immunol 147:3139-3144.
6. Lin YS, Yeh, T. M., Lin, C. F., Wan, S. W., Chuang, Y. C., Hsu, T. K., Liu HS, Liu, C. C., Anderson, R., Lei, H. Y. 2011. Molecular mimicry between virus and host and its implications for dengue disease pathogenesis. Exp Biol Med 236:515-523.
7. Huan Yang HW, Christopher J. Czura, and Kevin J. Tracey. 2008. The cytokine activity of HMGB1. J Leuko Bio 78:1-8.
8. Chen G, Ward MF, Sama AE, Wang H. 2004. Extracellular HMGB1 as a proinflammatory cytokine. J Interferon Cytokine Res: the official journal of the International Society for Interferon and Cytokine Research 24:329-333.
9. Allonso D, Belgrano FS, Calzada N, Guzman MG, Vazquez S, Mohana-Borges R. 2012. Elevated serum levels of high mobility group box 1 (HMGB1) protein in dengue-infected patients are associated with disease symptoms and secondary infection. J Clini Virol 55:214-219.
10. Allonso D, Vazquez S, Guzman MG, Mohana-Borges R. 2013. High mobility group box 1 protein as an auxiliary biomarker for dengue diagnosis. Am J Trop Med Hyg 88:506-509.
11. Blazquez AB, Escribano-Romero E, Merino-Ramos T, Saiz JC, Martin-Acebes MA. 2014. Stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy. Front Microbio 5:266.
12. Ron D, Walter P. 2007. Signal integration in the endoplasmic reticulum unfolded protein response. Nature Rev Mol Cell Bio 8:519-529.
13. Rebecca T. Marquez LX. 2012. Bcl-2:Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch. Am J Cancer Res. 2:214-212.
14. Levine B, Mizushima N, Virgin HW. 2011. Autophagy in immunity and inflammation. Nature 469:323-335.
15. Mizushima N. 2007. Autophagy: process and function. Genes Dev 21:2861-2873.
16. Dupont N, Jiang S, Pilli M, Ornatowski W, Bhattacharya D, Deretic V. 2011. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1beta. EMBO J 30:4701-4711.
17. Deretic V, Jiang S, Dupont N. 2012. Autophagy intersections with conventional and unconventional secretion in tissue development, remodeling and inflammation. Trends Cell Biol 22:397-406.
18. Richards AL, Jackson WT. 2013. How positive-strand RNA viruses benefit from autophagosome maturation. J Virol 87:9966-9972.
19. Lee YR, Lei HY, Liu MT, Wang JR, Chen SH, Jiang-Shieh YF, Lin YS, Yeh TM, Liu CC, Liu HS. 2008. Autophagic machinery activated by dengue virus enhances virus replication. Virology 374:240-248.
20. Mateo R, Nagamine CM, Spagnolo J, Mendez E, Rahe M, Gale M, Jr., Yuan J, Kirkegaard K. 2013. Inhibition of cellular autophagy deranges dengue virion maturation. J Virol 87:1312-1321.
21. Noisakran S, Onlamoon N, Hsiao HM, Clark KB, Villinger F, Ansari AA, Perng GC. 2012. Infection of bone marrow cells by dengue virus in vivo. Exp Hemato 40:250-259 e254.
22. Nakao S, Lai CJ, Young NS. 1989. Dengue virus, a flavivirus, propagates in human bone marrow progenitors. Blood 74:1235-1240.
23. Kristina B. Clark SN, Nattawat Onlamoon1, Hui-Mien Hsiao, John Roback, Francois Villinger, Aftab A. Ansari, Guey Chuen Perng. 2012. Multiploid CD61+ Cells Are the Pre-Dominant Cell Lineage Infected during Acute Dengue Virus Infection in Bone Marrow. PLoS One 7.
24. Tsai JJ, Liu LT, Chang K, Wang SH, Hsiao HM, Clark KB, Perng GC. 2012. The importance of hematopoietic progenitor cells in dengue. Ther Adv Hemato 3:59-71.
25. Pena J, Harris E. 2011. Dengue virus modulates the unfolded protein response in a time-dependent manner. J Biol Chem 286:14226-14236.
26. McLean JE, Wudzinska A, Datan E, Quaglino D, Zakeri Z. 2011. Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication. J Biol Chem 286:22147-22159.
27. Heaton NS, Randall G. 2010. Dengue virus-induced autophagy regulates lipid metabolism. Cell Host Microbe 8:422-432.
28. Perng GC. 2013. Role of Bone Marrow in Pathogenesis of Viral Infections. J Bone Marrow Res 1.
29. Chan LL, Shen D, Wilkinson AR, Patton W, Lai N, Chan E, Kuksin D, Lin B, Qiu J. 2012. A novel image-based cytometry method for autophagy detection in living cells. Autophagy 8:1371-1382.
30. Metz P, Chiramel A, Chatel-Chaix L, Alvisi G, Bankhead P, Mora-Rodriguez R, Long G, Hamacher-Brady A, Brady NR, Bartenschlager R. 2015. Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62. J Virol 89:8026-8041.
31. Mizushima N, Yoshimori T, Levine B. 2010. Methods in mammalian autophagy research. Cell 140:313-326.
32. Songane M, Kleinnijenhuis J, Netea MG, van Crevel R. 2012. The role of autophagy in host defence against Mycobacterium tuberculosis infection. Tuberculosis 92:388-396.
33. Lee YR, Hu HY, Kuo SH, Lei HY, Lin YS, Yeh TY, Liu CC, Liu HS. 2013. Dengue virus infection induces autophagy an in vivo study. J Biomed Sci 20.
34. Panyasrivanit M, Greenwood MP, Murphy D, Isidoro C, Auewarakul P, Smith DR. 2011. Induced autophagy reduces virus output in dengue infected monocytic cells. Virology 418:74-84.
35. Jiang S, Dupont N, Castillo EF, Deretic V. 2013. Secretory versus degradative autophagy: unconventional secretion of inflammatory mediators. J Innate Immun 5:471-479.
36. Sridharan A, Chen Q, Tang KF, Ooi EE, Hibberd ML, Chen J. 2013. Inhibition of megakaryocyte development in the bone marrow underlies dengue virus-induced thrombocytopenia in humanized mice. J Virol 87:11648-11658.
37. Basu A, Jain, P., Gangodkar, S. V., Shetty, S.,Ghosh, K. 2008. Dengue 2 virus inhibits in vitro megakaryocytic colony formation and induces apoptosis in thrombopoietin-inducible megakaryocytic differentiation from cord blood CD34+ cells. FEMS Immunol Med Mic 53:46-51.
38. Raslova H, Baccini V, Loussaief L, Comba B, Larghero J, Debili N, Vainchenker W. 2006. Mammalian target of rapamycin (mTOR) regulates both proliferation of megakaryocyte progenitors and late stages of megakaryocyte differentiation. Blood 107:2303-2310.
39. Noisakran S, Onlamoon N, Pattanapanyasat K, Hsiao HM, Songprakhon P, Angkasekwinai N, Chokephaibulkit K, Villinger F, Ansari AA, Perng GC. 2012. Role of CD61+ cells in thrombocytopenia of dengue patients. Int J Hematol 96:600-610.
40. Ogura M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, Hiroshi N, and Hidehiko S. 1985. Establishment of a Novel Human Megakaryoblastic Leukemia Cell Line, MEG-Ol, With Positive Philadelphia Chromosome Blood 66:1384-1392.
41. Lopez JJ, Palazzo A, Chaabane C, Albarran L, Polidano E, Lebozec K, Dally S, Nurden P, Enouf J, Debili N, Bobe R. 2013. Crucial Role for Endoplasmic Reticulum Stress During Megakaryocyte Maturation. Arterioscler Thromb Vasc Biol. 33:2750-2758.
42. Jin X, Ong SP, Lee LM, Leong YFI, Ng ML, Chu JJH. 2012. Dengue Virus Infection Mediates HMGB1 Release from Monocytes Involving PCAF Acetylase Complex and Induces Vascular Leakage in Endothelial Cells. PLoS One 7:e41932.