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研究生： 呂姿儀
Lu, Zi-Yi
論文名稱： 研究登革病毒非結構蛋白1在細胞凋亡之中的保護作用
Study on protective role of dengue non-structural protein 1 in cell apoptosis
指導教授： 張志鵬
Chang, Chih-Peng
學位類別： 碩士
Master
系所名稱： 醫學院 - 微生物及免疫學研究所
Department of Microbiology & Immunology
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 52
中文關鍵詞： 登革病毒 、Beclin-1 、自噬作用 、細胞凋亡 、登革非結構蛋白1
外文關鍵詞： Dengue virus, Beclin-1, autophagy, apoptosis, DENV non-structural protein 1
相關次數： 點閱：48  下載：2 
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    • 當黃熱病毒科的登革病毒感染人類時會引起嚴重的登革出血熱。而登革病毒的感染會誘導宿主多種的細胞反應，其中包括像是自噬作用和細胞凋亡。已知為了病毒的複製，登革病毒感染時會誘發細胞自噬作用來抵抗早期細胞凋亡的發生，然而其詳細究竟是透過甚麼機制尚未被完全釐清。另外越來越多的研究指出藉著調控相同的自噬蛋白能使自噬作用和細胞凋亡之間具有相互調控的現象。當自噬蛋白組成複合物時，便會活化自噬作用，而細胞得以存活。但相反地當細胞內的鈣蛋白酶（Calpains）或半胱天冬酶（Caspases）活化時，便會將自噬蛋白水解成片段，更進一步的反而會觸發細胞凋亡的發生。在篩選許多重要的自噬蛋白後，我們發現隨著登革病毒引起的細胞凋亡，同時有越來多BECN-1蛋白片段的產生。所以我們想知道在登革病毒的生命週期中，BECN-1是如何參與病毒誘導的自噬作用及細胞凋亡。首先，我們發現到當病毒感染缺乏BECN-1 的細胞時，登革病毒所引起的細胞凋亡會更加嚴重，進而抑制了病毒的複製。另一方面當登革病毒感染時加入泛半胱天冬酶抑制劑 (Z-VAD)， BECN-1蛋白片段產生的現象會受到抑制，此結果即表示了在登革病毒生命週期中半胱天冬酶調控了BECN-1 的水解。此外我們也發現到在感染登革病毒的情況下，登革非結構蛋白1 與BECN-1之間有相互作用。而當細胞表現登革非結構蛋白1時，會去促進細胞的自噬作用並抑制蕾莎瓦所引起的BECN-1片段的產生，同時也減少蕾莎瓦所誘發的細胞凋亡。而當細胞缺乏BECN-1 時，登革非結構蛋白1的保護作用就會消失。這些結果告訴我們，BECN-1與登革非結構蛋白1的作用可以維持細胞的生存。根據以上實驗結果，我們了解到BECN-1所調控的自噬作用與細胞凋亡的相互網絡，對於登革病毒複製週期是至關重要的； DENV-NS1具有保護BECN-1免於被切割的能力，並促進自噬作用來對抗早期的細胞凋亡。然而到了登革病毒的複製晚期，因為細胞內大量的CASPs活化，而將BECN-1切成蛋白碎片並誘導細胞凋亡的發生。

    Dengue virus (DENV) belongs to Flavivirade and causes severe dengue disease in humans, such as dengue hemorrhagic fever/dengue shock syndrome. DENV infection induces several cell responses, including autophagy and apoptosis. It has been known that DENV infection may trigger autophagy to against early apoptotic cell death and hence maintain viral replication; however, the underlying mechanisms are not fully understood. Growing studies have indicated that there are interplays between autophagy and apoptosis by regulation of the same autophagy-essential proteins. When these autophagy proteins are recruited to promote autophagy, the cells survive. In contrast, cell death signals activate cellular calpain (CAPN) or caspases (CASPs) to cleave these autophagy proteins into fragments, which further triggers apoptosis. After screening several essential autophagy proteins, we found that the fragmentation of BECN-1, a crucial protein of autophagy-initiating complex, was generated in DENV infection triggered apoptosis. Therefore, we went further to examine how BECN-1 participates in DENV-induced autophagy and apoptosis during viral life cycle. Loss of BECN-1 was found to promote apoptotic cell death and inhibit viral replication. Inhibition of CASPs by Z-VAD reduced the cleavage of BECN-1 in DENV-infecting cells, indicating a CASPs-mediated BECN-1 cleavage during DENV infection. We further found that DENV non-structural protein 1 (NS1) was co-localized and interacted with BECN-1 during DENV infection. Overexpression of NS1 reduced BECN-1 fragmentation and facilitated cellar autophagy to protect cells from sorafenib-induced apoptotic death. This cellular protection by NS1 was reduced in BECN-1-deficient cells, indicating that the interaction between BECN-1 and DENV-NS1 helps cell survival. In conclusion, we suggest that the regulation of BECN-1 is crucial for DENV replication. We reveal that DENV-NS1 preserves BECN-1 to promote autophagy function to antagonize early cell apoptosis; however, activated CASPs cleave BECN-1 into fragments to trigger apoptosis in the late stage infection.

    中文摘要 I Abstract II Acknowledgement III Table of contents VI -VI Abbreviations VII-VIII I. Introduction 1-8 I.1 Virology of DENV 1 I.1.1 Life cycle 1 I.1.2 The characteristics of viral components 2 I.2 Pathogenesis of DENV 3 I.3 Cell responses to DENV infection 4 I.3.1 DENV and autophagy 4 I.3.2 DENV and apoptosis 5 I.3.3 DENV and anti-apoptosis 6 I.4 The interplay between autophagy and apoptosis 7 I.4.1 Cross-regulation between autophagy and apoptosis 7 II. Objective and Specific Aims 9 III. Materials and methods 10-20 A. Materials 10-16 A-1 Cell lines 10 A-2. Viruses 10 A-3. Bacteria 10 A-4. shRNA plasmids 10 A-5. Antibody 11 A-6. Inhibitors 13 A-7. Reagents 13-16 B. Methods 17-20 B-1. Virus propagation 17 B-2. Plaque assay 17 B-3. DENV infection 18 B-4. Western blotting 18 B-5. Lentiviral-based RNAi gene knockdown 18 B-6. Lactate dehydrogenase (LDH) releasing assay 19 B-7. WST-1 assay 19 B-8. Immunofluorescence assay (IFA) 19 B-9. Cell death determination 19 B-10. Cell transfection 20 B-11. Co-immunoprecipitation 20 B-12. Statistical analysis 20 IV. Results 21-25 IV-1. Inhibition of autophagy enhances DENV2-induced apoptotic cell death 21 IV-2. DENV infection causes apoptosis and fragmentation of BECN-1 21 IV-3. BECN-1 protects cells from DENV -induced apoptosis 22 IV-4. CASPs mediate fragmentation of BECN-1 in DENV-infected cells 22 IV-5. BECN-1 interacts with DENV-NS1 in DENV-infected cells 24 IV-6. Sorafenib induces BECN-1 cleavage to increase apoptotic cell death in A549 cells 24 IV-7. DENV-NS1, but not DENV-NS3, attenuates BECN-1 fragmentation and promotes autophagy to inhibit Sorafenib-induced apoptotic cell death 25 V. Conclusion 26 VI. Discussion 27-31 VII. References 32-36 VIII. Figures 37-50 Figure 1. Inhibition of autophagy enhances DENV-induced apoptosis. 37 Figure 2. Increase of BECN-1 fragmentation and apoptosis in the late stage of DENV infection. 38 Figure 3. BECN-1 ablation promotes apoptotic cell death and inhibits virus production in DENV-infected cells. 39 Figure 4. The drug cytotoxicity and efficacy of CAPN inhibitor in A549 cells. 40 Figure 5. The CAPN inhibitor has no effects on cleavage of BECN-1, cell death and virus replication in the DENV-infected cells. 41 Figure 6. The drug cytotoxicity and efficacy of Z-VAD in A549 cells. 42 Figure 7. The CASPs mediate cleavage of BECN-1 in the DENV-infected cells. 43 Figure 8. The co-localization and interaction between BECN-1 and DENV-NS1 are found in DENV-infected cells. 44 Figure 9. Sorafenib, but not cisplatin or AR-12, induces BECN-1 cleavage and apoptotic cell death. 45 Figure 10. Overexpression of BECN-1 attenuates sorafenib-induced death. 46 Figure 11. Expression of DENV-NS1 attenuates BECN-1 fragmentation to facilitate cell autophagy and reduce sorafenib-induced apoptotic cell death. 47 Figure 12. Expression of DENV-NS3 is not able to reduce BECN-1 cleavage or sorafenib-induced cell death. 48 Figure 13. Knockdown of BECN-1 inhibits anti-apoptotic activity of DENV-NS1 in sorafenib-treated A549 cells. 49 Figure 14. The proposed schematic model of BECN-1 stability in regulating autophagy and apoptosis during DENV replication. 50 IX. Appendix 51-52 Appendix 1. The co-localization and interaction between BECN-1 and DENV-NS1 in Hela cells. 51 Appendix 2. Both CASP8 and CASP9 activates after DENV-2 infection. 52

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