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研究生：	吳鴻程 Wu, Horng-Cherng
論文名稱：	神經壞死病毒複製及死亡蛋白α誘發細胞死亡之分子機制 Molecular cell death mechanism in RGNNV infection: viral RNA replication and death protein α
指導教授：	吳金洌 Wu, Jen-Leih
共同指導教授:	洪健睿 Hong, Jiann-Ruey
學位類別：	博士 Doctor
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2010
畢業學年度：	98
語文別：	英文
論文頁數：	101
中文關鍵詞：	神經壞死病毒、細胞凋亡、後凋亡細胞壞死、粒線體膜電位、干擾性RNA 、蛋白α 、RNA複製
外文關鍵詞：	nervous necrosis virus, apoptosis, post-apoptotic necrosis, mitochondrial membrane potential, siRNA, protein α, RNA replication
相關次數：	點閱：205 下載：0
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石斑魚神經壞死病毒TN-1病毒株是從台灣病石斑魚體離出來，在分類上是屬於結病毒科，魚類結病毒屬，會感染許多高經濟魚種之稚幼魚，在石斑魚卵孵化後十幾天到吋苗期間，其死亡率可高達100%，造成水產養殖業重大的經濟損失。凋亡是細胞受到外界各式刺激而反應的自殺性死亡方式。在高等生物發育過程中，凋亡是經由基因調控，有秩序的淘汰贅餘細胞。當外來傷害刺激，如：放射線傷害、病毒感染或是致癌基因，也會誘發凋亡，將受損細胞清除，以保護生物體的安全。本篇論文主要目的是探討神經壞死病毒之死亡蛋白α和病毒基因體複製誘發細胞死亡的分子機制。本篇論文分為二部份：(1)確定死亡蛋白α誘發宿主細胞死亡途徑與(2)研究病毒基因體複製與誘發宿主細胞死亡的關係。
論文第一部份研究顯示，神經壞死病毒死亡蛋白α能誘發石斑魚GF-1細胞進行凋亡及後凋亡細胞壞死現象(第二章)。經由Annexin-Ⅴ染色及TUNEL分析，證實死亡蛋白α所誘導之早期及中期的細胞凋亡特徵。以吖啶橙(Acridine orange)和溴化乙錠(Ethidium bromide)進行複染，證實蛋白α 誘導的後凋亡細胞壞死現象。死亡蛋白α亦會促使粒線體膜電位流失，進而造成細胞色素(cytochrome) c從粒線體釋出至細胞質，因而證實蛋白α 與粒線體功能之消失有關。抗凋亡蛋白zfBcl-xL能抑制蛋白α引發的粒線體膜電位流失，然而若以RNA干擾技術減少病毒死亡蛋白α表現，只能部份抑制病毒誘發之細胞凋亡現象。
論文第二部份研究神經壞死病毒誘發細胞凋亡，需要新合作蛋白的參與(第三章)。當以紫外線照射病毒，破壞病毒複製能力後，若只有病毒顆粒吸附在細胞表面，無法誘發細胞凋亡現象，證明神經壞死病毒誘發細胞凋亡不是經由外在接受器誘發凋亡的途徑。以RNA干擾技術減少病毒RNA複製酶(蛋白質Ａ)表現，可抑制病毒基因體複製及死亡蛋白α及B2的表現，進而抑制細胞凋亡及粒線體死亡訊息傳導，增加細胞病毒感染後的存活率。綜合所述，神經壞死病毒誘發粒線體失調的凋亡現象，是經由內部誘發途徑，且必需經由病毒基因體複製及死亡蛋白基因表現。本論文藉由探討死亡蛋白α和病毒基因體複製誘發細胞死亡的分子機制，對神經壞死病毒有更寛廣的瞭解，希望末來能應用於該病毒的檢測、防疫及治療，為台灣水產養殖業，略盡微薄心力。

The red spotted grouper nervous necrosis virus (RGNNV) TN1 strain was isolated from grouper in Taiwan. RGNNV belongs to betanodavirus, and it predominantly infects a wide variety of larval and juvenile marine fish species in worldwide, causing severe morbidity and mortality and significant economic losses to the aquaculture industry. Apoptosis is a suicidal process whereby individual cells of multicellular organisms in response to a wide variety of stimuli. The process is genetically controlled and preprogrammed to eliminate redundant cells during development, and is used as an emergency response following radiation damage, viral infection, or aberrant cell growth induced by oncogenes. The aim of this dissertation is to study the molecular cell death mechanism induced by RGNNV infection. Two parts of studies are included: (1) to identify viral death inducer proteinα induced cell death mechanism, and (2) to identify why RGNNV replication is required for triggering host cell death.
The first part of this dissertation showed that protein α can induce apoptosis and post-apoptotic necrosis in GF-1 cells (in chapter 2). Apoptotic features were identified either by Annexing V-FLUOS staining or by TUNEL assay, and then post-apoptotic necrotic cells were identified by alcidine orange/anthodium bromide dual dye staining. Further, protein α induced progressive loss of mitochondrial membrane potential (MMP) that its death is correlated with cytochrome c release and blocked by anti-apoptotic protein zfBcl-xL in GF-1 cells. Furthermore, the knockdown of protein α expression by its specific siRNA partially inhibited necrotic cell death with RGNNV infection.
In second part, we proposed that the newly synthesized protein-dependent pathway is required for RGNNV-induced cell death (in chapter 3). First, UV irradiation of the virus could effectively block viral replication and cell death that RGNNV-induced cellular death is required some death factors for triggering this death pathway. Then, we try to block protein A expression by its specific siRNA, in the results of knockdown of protein A, completely blocked viral genomic replication and blocked the expression of the viral death inducers including proteins α and B2 which is correlated to prevent the phosphatidylserine exposure, mitochondria-mediated death signaling, and enhancing cellular viability. Our results suggest that RGNNV-induced necrotic cell death is via newly synthesized protein dependent-pathway such as viral death inducers protein α and protein B2 for triggering mitochondria-mediated death pathway, but caspase-independent pathway. These findings may provide new insights into RNA virus-induced host pathogenesis and clinical treatment.

Abstract I 
中文摘要 III 
Acknowledgement V 
Contents VI 
Abbreviation List X 
Chapter 1 Overview introduction 1 
1.1 Nervous necrosis virus (NNV) 2 
1.1.1 History of viral nervous necrosis disease 2 
1.1.2 Geographic and Species distribution 3 
1.1.3 Clinical signs 3 
1.1.4 Histopathology 4 
1.1.5 The Molecular biology of NNV 4 
1.1.6 Taxonomy of NNV 5 
1.2 Cell death 5 
1.2.1 Apoptosis 7 
1.2.2 Necrosis 9 
1.3 Objective and Specific aims 13 
1.4 Figures 16 
Figure 1.1 Schematic models of pathways leading to cell death 16 
Figure 1.2. Pathways and markers of necrosis and apoptosis (Adapted from ref. 22). 17 
Figure 1.3. Three major apoptotic pathways (Adapted from ref. 27). 17 
Figure 1.4. Extrinsic and intrinsic apoptotic pathways (Adapted from ref. 31) 18 
Figure 1.5. Signal transduction cascades leading to necrotic cell death and the role of RIP1 (Adapted from ref. 41) 18 
Figure 1.6. Phase-contrast fluorescence image of RGNNV TN1-infected GL-av cells. 19 
Chapter 2 Zebrafish anti-apoptotic protein zfBcl-xL can block betanodavirus protein α-induced mitochondria-mediated secondary necrosis cell death 20 
Abstract 21 
2.1 Introduction 22 
2.2 Materials and methods 24 
2.2.1 Cell culture and reagents 24 
2.2.2 Cloning of RNA2 and construction of expression vector 24 
2.2.3 Western blot analysis 25 
2.2.4 Apoptotic cells and secondary necrotic cells assay 26 
2.2.5 Evaluation of MMP 28 
2.2.6 Preparation of mitochondria from RGNNV RNA2-transfected cells 28 
2.2.7 Cell counts and statistical analyses 29 
2.2.8 Caspase activity assays 29 
2.2.9 Construction and selection of GF-1 cells expressing RGNNV RNA2-specific siRNA 30 
2.3 Results 32 
2.3.1 Cloning of RGNNV RNA2 32 
2.3.2 Viral protein expression in GF-1 cells 32 
2.3.3 Influence of protein α on post-apoptotic necrosis cell death 32 
2.3.4 Influence of protein α on GF-1 MMP and cytochrome c release 33 
2.3.5 Effect of anti-apoptotic protein zfBcl-xL on protein α-induced loss of GF-1 MMP 34 
2.3.6 Knockdown of protein α by siRNA partially inhibited necrotic cell death following RGNNV infection 35 
2.4 Discussion 37 
2.5 Tables and figures 39 
Table 2.1. The siRNA sequences are sense strand of RNA2 in this study 39 
Table 2.2. The influence of viral titers with knockdown of RNA2 by Its specific siRNA at different post-infection timea 39 
Figure 2.1. Identification of viral protein expression in GF-1 cells. 41 
Figure 2.2. Identification of RGNNV RNA2 induces apoptotic and post-apoptotic necrosis in GF-1 cells. 44 
Figure 2.3 RGNNV RNA2-transfection induces loss of MMP in GF-1 cells. 46 
Figure 2.4. Anti-apoptotic protein zfBcl-xL blocks RNA2-induced loss of MMP in GF-1 cells. 48 
Figure 2.5. Effect of protein α knockdown by siRNA on the necrotic cell death. 49 
Chapter 3 RGNNV induces mitochondria-mediated cell death via newly synthesized protein-dependent pathway in fish cells 50 
Abstract 51 
3.1 Introduction 52 
3.2 Materials and Methods 55 
3.2.1 Cell line and virus 55 
3.2.2 UV-irradiated virus 55 
3.2.3 Cloning and sequence analysis of NNV RNA1 55 
3.2.4 Expression of recombinant protein A 56 
3.2.5 Preparation of a polyclonal antibody against protein A 57 
3.2.6 Identification of RGNNV protein A by immunostaining 58 
3.2.7 Construction and selection of GF-1 cells expressing RGNNV RNA1-specific siRNA 58 
3.2.8 Total mRNA preparation 59 
3.2.9 Northern blot analysis 60 
3.2.10 Reverse transcriptase-polymerase chain reaction 60 
3.2.11 Western blot analyses 60 
3.2.12 Annexin-V–FLUOS staining 61 
3.2.13 Quantification of cell viability 61 
3.2.14 Evaluation of mitochondrial membrane potential 62 
3.2.15 Preparation of mitochondrial cytochrome c 62 
3.2.16 Caspase-3 activation assay 63 
3.2.17 Cell counts and statistical analyses 63 
3.2.18 Dual staining of necrotic cells by ethidium bromide and acridine orange dyes 63 
3.3 Results 65 
3.3.1 RGNNV-induced necrotic cell death is via a newly synthesized viral proteins dependent pathway 65 
3.3.2 Cloning of protein A and its localization 66 
3.3.3 Knockdown of RdRp by siRNA can block viral RNA replication and viral protein expression for reducing host cell death 67 
3.3.4 Reduced viral proteins effectively prevents mitochondria-mediated necrotic cell death 68 
3.4 Discussion 70 
3.5 Tables and figures 72 
Table 3.1. The siRNA sequences are sense strand of RNA1 in this study 72 
Table 3.2. The siRNA and actin sequence primers for stable expressing RNA1 siRNA. 72 
Table 3.3. The influence of viral titers with knockdown of protein A by its specific siRNA at different post-infection timea 72 
Figure 3.1. Effects of UV irradiation of RGNNV on RNA replication in fish cells. 74 
Figure 3.2. Cloning and localization of RGNNV protein A in fish liver cells. 77 
Figure 3.3. Identification of RNA1-specific siRNA-producing cell lines. 79 
Figure 3.4. Effect of protein A expression on viral RNA replication and viral protein expression. 80 
Figure 3.5. Blockage of viral protein expression via knockdown of protein A can prevent necrotic cell death and enhance cellular viability. 82 
Figure 3.6. Influence of viral protein expression on mitochondria-mediated cytochrome c release 84 
Chapter 4 Conclusion and future perspectives 85 
4.1 Conclusion 86 
4.2 Future perspectives: 87 
4.2.1 siRNA therapy of viral nervous necrosis 87 
4.2.2 The down-stream factors of RGNNV induced mitochondria-mediated cell death 87 
References 89 
Publications 101
                                    

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