登革病毒（Dengue virus; DENV）每年導致超過3.9億人感染。登革病毒感染會導致輕度登革熱（DF）、嚴重登革熱出血熱（DHF）以及登革熱休克綜合徵（DSS），甚至死亡。登革病毒是單鍊正股核醣核酸包膜病毒，其基因是由三個結構蛋白；核蛋白（C），前膜蛋白（prM / M），包膜蛋白（E）和七個非結構蛋白（NS1至NS5）組成，這些病毒蛋白各自扮演了從病毒RNA複製到蛋白質合成等不同的角色。先前的研究指出登革病毒感染會誘導自噬現象(autophagy)並幫助病毒複製。他人報導登革病毒感染會觸發自噬相關囊泡的釋放並藉此逃避免疫系統監測。自噬現象也負責病原體和細胞內許多蛋白的運送，包括高遷移率族蛋白B1（HMGB1），並可分為選擇性和非選擇性兩種降解機制。本研究闡明了自噬體(autophagosome)，HMGB1和登革病毒蛋白之間的關係，包括登革病毒蛋白是否可以選擇性的被自噬體包裹及分泌型自噬現象是否參與病毒蛋白質的釋放。我們首先確認登革病毒的感染可以誘導自噬現象在肺癌細胞(A549)發生。接著從登革病毒感染的肝癌細胞和肺癌細胞中純化出自噬體，利用電子顯微鏡觀察確認受免疫金標記的C，NS1和NS3蛋白存在其中。我們還證明了登革病毒的C，E，NS1，NS3，NS4B和HMGB1蛋白可以選擇性的被包裹到自噬體中。此外登革病毒的C，E，NS1和NS3蛋白的表現會隨著感染時間逐漸增加，顯示這些蛋白質並沒有被自噬體降解。進一步使用溶酶體融合的抑制劑(CQ)抑制自噬體降解發生，結果顯示在抑制劑作用的細胞中登革蛋白C，E和NS1的表現相對於LC3蛋白要來的低，有可能在CQ處理下分泌型自噬反應被誘發並進一步協助這三種蛋白之釋放。HMGB1在CQ作用下釋放量的增加支持了我們的推測。此外我們也證實純化的自噬體中不含有病毒顆粒但仍具有感染性。總而言之，本研究證實在登革病毒的感染下，若自噬降解反應被抑制時可以誘導分泌型自噬的發生並將登革蛋白C、E和NS1以及HMGB1釋放到細胞外。

Dengue virus (DENV) causes over 390 million people infection every year. Dengue disease ranges from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and death. DENV is an enveloped, single-stranded, positive-sense RNA virus. DENV genome consists of three structural proteins capsid protein (C), premembrane/ membrane protein (prM/M), envelope protein (E) and seven nonstructural proteins (NS1 to NS5). These proteins play diverse roles from viral RNA replication to protein synthesis. We previously reported that DENV infection induces autophagy to enhance virus replication. Others further reported that DENV infection triggered autophagy related vesicle releasing to evade immune system surveillance. Autophagic machinery is involved in selective and non-selective cargoes degradation process and participates in extracellular delivery of a number of pathogen particles and cytosolic proteins, such as high mobility group box 1 (HMGB1), which was significantly increased in DENV-infected patients. This study clarified the relationship among autophagosome, HMGB1 and DENV proteins, including which DENV-protein can be selectively recruited to the autophagosome and whether secretory autophagy is involved in the release of these recruited proteins. Initially, we showed that autophagy is induced by DENV2 infection of lung cancer A549 cells. We then purified the autophagosome from DENV2-infected Huh7 cell and A549 cells. We further reveal that DENV-C, E, NS1, NS3, NS4B and HMGB1 proteins are existed in the purified autophagosomes by Western blotting. We confirmed that C, NS1 and NS3 proteins are indeed in the autophagosome by immunogold labeling transmission electron microscopy. We also clarified that DENV-C, E, NS1, NS3, NS4B and HMGB1 can be selectively recruited to the autophagosome. Furthermore, the levels of C, E, NS1 and NS3 proteins increased alone with infection time, indicating that these proteins were not degraded. We further used chloroquine (CQ) to block lysosome fusion at 48 h.p.i. The levels of DENV-C, E and NS1 proteins decreased compared to the LC3-II protein levels in DENV2 infected A549 cells, indicating that an unconventional secretory autophagy was triggered, which leads to the exocytosis of these three proteins under CQ treatment conditions. The increase release of HMGB1 after CQ treatment, support our speculation that secretory autophagy was promoted when degradative autophagic process was inhibition. In conclusion, this study provide the evidence that an unconventional secretory autophagy can be induced during under DENV infection when autophagy degradation was suppression.

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