臺灣博碩士論文加值系統

登革熱病毒感染會誘導自嗜體的產生，且此反應已被證實會增加病毒的感染力，被認為登革熱病毒不僅可以逃避自嗜反應還可以進一步的去利用它幫助自己產生子代，但關於登革熱病毒與自嗜體詳細的交互作用，目前並不明確。在本篇研究我們發現，加入自嗜抑制劑3-methyladenine(3-MA)與自嗜催化劑Rapamycin會造成病毒的總產量的增加，但與病毒的複製和轉譯並無直接關係，卻會造成病毒分布位置的改變，所以我們推測自嗜作用會影響病毒的運輸。在此為了更進一步研究病毒感染初期病毒的運輸途徑，我們使用了單病毒追蹤技術，這是一個高敏感度高解析率的工具，可以藉此去研究細胞內囊泡的運輸。我們首先使用共軛焦顯微鏡去確認細胞內螢光蛋白標定的囊泡與自嗜體的疊合情形，並使用親脂性的染劑去標定病毒，發現不論是早期還是晚期核內體都會與病毒和自嗜體相疊合，我們實際去追蹤登革熱病毒發現病毒會藉由早期核內體傳輸到自嗜體，也會藉由晚期核內體傳輸到自嗜體，這揭露了病毒有多重運輸途徑的可能。最後，在加了核內體的抑制劑可以有效的抑制病毒與自嗜體疊合的現象，在加了自嗜體的抑制劑則會使病毒與內涵體的疊合率增加，這雙重證明了我們的假說。在本篇實驗我們除了證明自嗜反應會直接影響病毒的運輸，且發現此運輸是由早期或晚期核內體傳輸到自嗜體，我們認為這可能揭露病毒有多重運輸途徑的可能性。

Dengue virus (DENV) infection induces autophagy which increases the virus production, suggested that DENV can escape from autophagy degradation that may be used for virus production. However, the interaction between DENV and autophagy has not been revealed comprehensively. The data in our research showed that autophagy strongly influences DENV production, replication and translation. Moreover, we would like to examine DENV intracellular transport and to understand the relationships between DENV transport and autophagy. Single-virus particle tracking technology allows us to investigate the dynamic interactions between individual virus particle and cellular factors. In our research, we have successfully tracked a single DENV particle with lipophilic fluorescence dye DiD in living Huh-7.5-GFP-LC3 cells which harbor various fluorescence-fused tags to indicate different cellular components using a home-built real-time multi-fluorescence microscopy. The tracking images showed that DiD-labeled DENV particles entered endosomes by observing colocalization with RAB5 and RAB7. Subsequently, we also found out that the endosomes enclosing the DENV progress fused with LC3-positive autophagosomes. In summary, we revealed that the DENV particles were transported from endosome to autophagosome during the early stage of viral infection.

目 錄
中文摘要……………………………………………………………………………………………………….…… i
英文摘要…………………………………………………………………………………………………………….. ii
目錄…………………………………………………………………………………………………………………... iii
圖目錄………………………………………………………………………………………………………………. v
第一章 前言…………………………………………………………… 1
1.1 Dengue virus……………………………………………………………………..……………..… 1
1.2 DENV vesicular-transport pathway…………………………………………………… 2
1.4 Live-cell, real-time fluorescent imaging techniques…………………..………… 3
1.5 Single-virus tracking……………………..……………………………………………….…… 4
1.6 Autophagy……………………………………………………………………………………….….. 5
第二章 材料與方法………………………………………………………………….. 7
2.1 Cell cultures…………………………………………………………………………………………. 7
2.2 DENV amplification and infection………………………………………………..……… 7
2.3 Transfection……………………………………………………………………………………….… 7
2.4 Immunofluorescence……………………………………………………………………….…… 8
2.5 Western blot analysis……………………………………………………………………………. 8
2.6 Plaque assay…………………………………………………………………………………….….. 9
2.7 DiD labeling DENV particles………………………………………………………………. 9
2.8 Fluorescence confocal microscopy imaging……………………………………….. 10
2.9 Time-lapse dual-channel fluorescence microscopy system…………………. 10
第三章 研究動機與目的………………………………………………………………….. 12
第四章 結果…………………………………………………………………………………… 13
4.1 Relationship between autophagy and dengue virus…..... …..... …..……....... 13
4.2 Dengue virus particles colocalization behavior ..………………………………….. 16
4.3 Dengue virus transport pathway……………………………………………………………. 19
4.4 Effect of movement inhibitor………………………………………………………………… 21
第五章 討論…………………………………………………………… 27
第六章 參考文獻…………………………………………………………… 31
第七章 圖表…………………………………………………………… 36

圖表目錄
Figure 1. DENV induces autophagy which promote virus production.................. 36 Figure 2. DENV induces autophagy which promote virus replication.................. 38 Figure 3. DENV induces autophagy which promote virus translation.................. 40 Figure 4. Dengue virus labeling.............................................................. 42 Figure 5. The effects of 3-methyladenine (3-MA) and rapamycin on the distribution of DENV particles................................................................................. 44 Figure 6. Co-localization of DENV particles with early endosomes and autophagosomes................................................................................. 46 Figure 7. Co-localization of DENV particles with early endosomes and autophagosomes................................................................................ 48 Figure 8. Co-localization of DENV particles with late endosomes and autophagosomes. ............................................................................... 50 Figure 9. Co-localization of DENV particles with late endosomes and autophagosomes. ............................................................................... 52 Figure 10. Setup for real time multi-fluorescence microscopy. ....................... 54 Figure 11. The translocation of a single DENV particle from early endosome to autophagosome. ................................................................................ 56 Figure 12. The translocation of a single DENV particle from late endosome to autophagosome. ................................................................................ 58 Figure 13. The speeds analyze of a single DENV particle. ............................. 60 Figure 14. TIP decreases DENV transport to autophagosomes. ............................................................................... 62 Figure 15. TIP decreases DENV transport to
autophagosomes. ............................................................................... 65 Figure 16. Endosome dominant negative (DN) RAB7-Q67L decreases DENV transport to autophagosomes. ................................................................ 68 Figure 17. 3-MA decreases the effect of DENV transport from early endosome to autophagosome. ................................................................................ 71 Figure 18. 3-MA decreases the effect of DENV transport from late endosome to autophagosome. ............................................................................... 73

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