臺灣博碩士論文加值系統

登革病毒是由蚊子所傳播的病原體之一，目前全世界已有超過一百個國家流行登革病毒所引起的疾病。在臨床上，登革病毒感染人類所引起的疾病甚廣，但是目前對於宿主與病毒間的相互作用以及病毒的生命週期尚不清楚，因此並沒有抗病毒藥物可以治療。病毒需要仰賴宿主細胞的能量來幫助其增殖，粒線體(mitochondria)是細胞內產生能量的地方且扮演著氧化代謝的角色。此外，粒線體是高度動態平衡的胞器。最近的研究發現登革病毒在感染後期階段(24至72小時)會造成粒線體形態改變。雖然觀察時間點皆在病毒蛋白轉譯完成的階段，所觀察到粒線體形態的變化卻有衝突的結果。然而我們實驗室過去研究發現登革病毒感染後的一個小時即可引起細胞自噬反應(autophagy)。因此我們認為在病毒感染初期所引起的自噬反應會對粒線體造成影響。為了驗證這個假說，我利用活細胞影像技術來觀察病毒感染初期對於粒線體形態的影響，結果顯示在病毒感染後三個小時會使粒線體呈現片段的現象，利用粒線體形態分析軟體量化後，結果顯示在感染後三個小時會使形態中fragmentation的比例增加而elongation的比例減少。此外，加入自噬作用的抑制劑於病毒感染的細胞，顯示抑制自噬作用可以保護病毒所引起粒線體的片段化。我進一步測定病毒感染後對於粒線體膜電位與質量的變化，結果顯示病毒感染初期會降低粒線體膜電位且會使質量下降。然而，加入自噬作用的抑制劑於病毒感染的細胞內，其膜電位降低的發生時間會受到延遲且病毒所導致的質量減少則會恢復。這些結果顯示自噬作用參與在調節粒線體膜電位的改變以及粒線體的生合成之中。因此本篇研究證明在感染初期階段登革病毒可以透過自噬作用的訊號來調節粒線體的動態平衡。

Dengue virus (DENV) is a mosquito-borne pathogen which presents in over 100 countries. DENV infection causes a wide range of clinical symptoms but no approved antiviral drug is available currently due to little known about virus-host interaction and life cycle of DENV. Virus relies on host cell metabolism for its proliferation. Mitochondria are highly dynamic organelles that regulate cell metabolism. Recent studies found that DENV induces mitochondrial morphology alteration at the late stage of infection, probably at 24, 48 and 72 h post-infection. These studies showed conflict results even though the observation time were the same at late infectious stage. Our previous study showed that DENV induces host cellular autophagy at 60 min post-infection. It raises a question that autophagy may participate in the alteration of mitochondria at early infectious stage. To investigate the possibility, I used live-cell imaging technique to observe mitochondrial morphology in DENV-infected cells. The results showed that mitochondrial morphology became fragmented at 3 h post-infection. Quantitative analysis found that fragmented population increased in DENV-infected cells. Moreover, autophagy inhibitors prevent the DENV-induced mitochondrial fragmentation. Mitochondrial membrane potential and mass were decreased in DENV-infected cells at early infectious stage. Interestingly, when co-treated with DENV and autophagy inhibitor, mitochondrial membrane potential disruption was delayed, and the mass recovered as basal level. These results suggest that autophagy participates in the disruption of mitochondrial membrane potential and the synthesis of mitochondrial mass during DENV infection. This study demonstrated that DENV-affected mitochondria is an autophagy-dependent event at early infectious stage.

中文摘要 i
Abstract ii
Contents iii
Introduction 1
1. Dengue virus 1
2. Dengue virus life cycle 3
3. Mitochondria 4
4. Autophagy 7
Hypothesis and Specific Aims 10
Materials and Methods 11
1. Cell culture 11
2. DENV amplification 12
3. Plaque assay 12
4. Virus condensation 13
5. Transfection 14
6. Fluorescence confocal microscope image 14
7. Mitochondrial membrane potential detection 15
8. Mitochondrial mass detection 15
9. MicroP software 2011 version 1.1.3a- mitochondrial morphology quantification 16
Results 18
1. DENV induces mitochondria fragmentation at 3 h post-infection 18
2. Fragmented mitochondria increase at 3 h post-infection 18
3. Mitochondria become fragmented at early stage of infection and return to basal level at the late infectious stage 19
4. Inhibition of autophagy blocks DENV-induced mitochondrial fragmentation 20
5. Mitochondrial dynamics is autophagy-dependent at early infectious stage 21
6. Autophagy participates in disruption of mitochondrial membrane potential 21
7. Autophagy regulates mitochondrial mass 22
8. Mitochondria of Huh7.5 cells are fragmented at 3 h post-infection 22
9. Mitochondria of Huh7.5 cells become fragmented at early stage of infection and return to basal level at the late infectious stage 24
10. Autophagy alters mitochondria dynamics at early infectious stage 24
11. Autophagy regulates mitochondrial membrane potential 25
12. Autophagy mediates mitochondrial mass 25
Discussion 27
References 30
Figures and legends 37

Figure 1. DENV induces mitochondria fragmentation at 3 h post-infection 37
Figure 2. Mitochondrial skeleton becomes shorter at 3 h post-infection 38
Figure 3. Fragmented mitochondria increase at 3 h post-infection 39
Figure 4. Mitochondria become fragmented at early stage of infection and return to basal level at the late infectious stage 40
Figure 5. 3-MA inhibits mitochondrial fragmentation 41
Figure 6. CQ protects mitochondrial fragmentation 42
Figure 7. 3-MA affects mitochondrial dynamics at early infectious stage 43
Figure 8. CQ affects mitochondrial dynamics at early infectious stage 44
Figure 9. Autophagy regulates mitochondrial membrane potential 45
Figure 10. Autophagy regulates mitochondrial mass 46
Figure 11. Mitochondria of Huh7.5 cells show fragmented at 3 h post-infection 47
Figure 12. Mitochondrial skeleton become shorter at 3 h post-infection 48
Figure 13. Fragmented mitochondria increase in Huh7.5 cells 49
Figure 14. Mitochondria of Huh7.5 cells become fragmented at early stage of infection and return to basal level at the late infectious stage 50
Figure 15. Beclin 1 knockdown prevents mitochondrial fragmentation 51
Figure 16. Autophagy inhibition delays mitochondrial membrane potential disruption 52
Figure 17. Autophagy mediates mitochondrial mass 53

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