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研究生：	巫瑞旭 Reshi, Latif
論文名稱：	石斑魚虹膜病毒（GSIV）之絲氨酸蘇氨酸激酶基因誘導石斑魚細胞死亡之分子機制 The molecular cell death mechanism induced by grouper sea perch iridovirus (GSIV) serine theroine kinase gene in grouper fish cells
指導教授：	王浩文 Wang, Hao-Ven
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 - 生命科學系 Department of Life Sciences
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	149
外文關鍵詞：	GSIV, apoptosis, necrosis, mitochondria, pathogenesis, host, iridoviridae, virus
相關次數：	點閱：63 下載：1
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Abstract.

Aquaculture is one of the most important economic activities in Asia and is presently the fastest growing sector of food production in the world. Explosive increases in global fish farming have been accompanied by an increase in viral diseases. Viral infections are responsible for huge economic losses in fish farming, and control of these viral diseases in aquaculture remains a serious challenge. The prevention and treatment of viral infections are
particularly challenging because viruses use the host cell machinery to replicate, and interrupting viral replication without damaging host cell structures or processes presents a puzzle. Thus, the development of antiviral vaccines or drugs requires an understanding of both the host immune system genetics and viral gene function.
Apoptosis plays a central role in the normal development and homeostasis of multicellular organisms. The process of apoptosis is controlled by a range of cell signaling pathways that originate either from cells external environment or from within the cell itself.
The cell’s fate to undergo apoptosis depends mainly on the dynamic balance between the Bcl-2 family sensor proteins, which both promote and inhibit apoptosis. In many (but not all) cases,these proteins appear to influence the activation of caspase family members (proteases), which
perform the ‘‘execution’’ phase of apoptosis by cleaving a number of cellular proteins and bringing about the destruction of cellular structures. Apoptosis may be used by the host to limit the production of viruses or to disseminate them. However, viruses use the apoptosis process
to produce sufficient virus progeny to facilitate virus release.
The megalocytiviruses belonging to family iridoviridae infect a wide range of tropical marine and freshwater fish, including grouper, gourami, cichlid, red sea bream, angel
fish, sea bass and lamp eye, causing similar diseases in each species. The forms of cell death evoked by iridoviruses are continually being disclosed, and they include typical apoptosis and non-apoptotic cell death. However, the signaling pathways involved in these processes remain largely unknown. In this study, i showed that GSIV encoded serine/threonine kinase gene induces apoptotic cell death via a p53-mediated upregulation of Bax which causes a loss of MMP. Therefore we first time reported that the tumor suppressor protein p53 is involved to propagate the signal to commit suicide in fish cells. This loss then mediates cell death signaling, which results in an activation of the caspase-mediated cell death pathway at the mid-to-late stages of viral replication. These findings may provide new insight into GSIV-induced pathogenesis.
In 2nd part of my study, overexpression of Bcl-2 and Bcl-xL anti-apoptotic genes protect GF-1 cells from GSIV encoded serine/threonine kinase induced pro-apoptotic stimuli.In this study, i try to determine if these proteins could provide equal protection when challenged with a viral apoptotic gene. Bcl-2 and Bcl-xL overexpression inhibit Bax oligomerization and mitochondrial outer membrane permeabilization. Therefore we demonstrated that GSIV st-kinase induced cell death is prevented by the antiapoptotic Bcl-2 and Bcl-xL, which enhances host cell viability through blockage of mitochondrial disruption and caspase 9 and caspase 3 activation. These results indicate that Bcl-xL and Bcl-2 confer different ability to protect against GSIV serine/threonine kinase induced cell death which appears to be dependent on the molecular mechanism.

Abstract……………………………………………………………………………………..i-ii
Chapter 1 .................................................................................................................................. 1
1 Introduction .......................................................................................................................... 2
2 Apoptosis ............................................................................................................................. 2
3 Necrotic Cell Death.............................................................................................................. 4
4 Apoptosis in Virus Infected Cells ........................................................................................ 4
5 ROS friend and foe of cells .................................................................................................. 6
5.1 Mitochondria: Source and target of ROS.......................................................................... 8
5.2 ROS in Endoplasmic Reticulum. ...................................................................................... 9
6 Iridoviruses .......................................................................................................................... 9
6.1 Different molecular cell death mechanisms induced by Iridoviruses .............................10
6.1.1 Largemouth bass virus ................................................................................................. 10
6.1.2 Singapore Grouper iridovirus ...................................................................................... 12
6.2 Megalocytivirus strain .................................................................................................... 15
6.2.1 Rock bream iridovirus.................................................................................................. 15
6.2.2 Grouper sea perch Iridovirus induced cell death. ........................................................ 17
7 Conclusion: ........................................................................................................................ 27
8 References. ......................................................................................................................... 30
Chapter 2 ................................................................................................................................ 42
GSIV serine/threonine kinase induces apoptotic cell death via p53 and pro-apoptotic gene
Bax up regulation in fish cells..................................................................................................42
1 Abstract .............................................................................................................................. 43
2 Introduction: ....................................................................................................................... 45
3 Material and methods. ........................................................................................................ 48
v
3.1 Chemicals, drugs and antibodies used: ........................................................................... 48
3.2 Drug treatment. ............................................................................................................... 48
3.3 Cell culture: ..................................................................................................................... 48
3.4 Viruses ............................................................................................................................ 48
3.5 Virus Infection ................................................................................................................ 49
3.6 RNA isolation and RT-PCR Cloning:............................................................................. 49
3.7 Sequence analysis of GSIV Kinase ................................................................................. 50
3.8 EGFP ST kinase fusion gene construction...................................................................... 50
3.9 Transition transfection Assay ......................................................................................... 50
3.10 Analysis of apoptosis .................................................................................................... 51
3.10.1 TUNEL assay ............................................................................................................. 51
3.10.2 Hoechst 33258 nuclear staining .............................................................................. 52
3.11 Protein extraction and western blotting analysis .......................................................... 53
3.12 Evaluation of mitochondrial membrane potential (MMP) with a Lipophilic cationic
dye ............................................................................................................................................ 54
3.13 Cell counts and statical analysis ................................................................................... 54
4 Results. .............................................................................................................................. 55
4.1 GSIV ST kinase gene expression profile and cloning .................................................... 55
4.2 Location of Full-Length Kinase Protein in GF1 Cells. ................................................... 55
4.3 Overexpression of GSIV ST kinase induces apoptosis, or cell death .............................56
4.3.1 Typical apoptosis on the basis of morphological changes. .......................................... 56
4.3.2 Expression of GSIV Kinase Induces Typical Apoptosis Phenomena Such as Nuclear /
chromatin condensation ........................................................................................................... 57
4.3.3 Expression of GSIV Kinase Induces Typical Apoptosis Phenomena Such as DNA
Fragmentation .......................................................................................................................... 57
4.4 Overexpression of GSIV ST kinase upregulate p53, Bax and down regulate Bcl-2
protein in GF-1 cells. ............................................................................................................... 57
4.5 ST kinase overexpression can induce MMP loss in GF-1 cells ...................................... 58
vi
4.6 ST kinase overexpression can activate caspases cascade (-9 and -3) ............................. 59
4.7 P53 activity inhibitor, pifithrin-ά can reduce the ST kinase-induced cell death............. 59
5 Discussion .......................................................................................................................... 61
6 References .......................................................................................................................... 93
Chapter 3. ............................................................................................................................... 98
Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine
kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells ..................................... 98
1 Abstract .............................................................................................................................. 99
2 Introduction ...................................................................................................................... 100
3. Material and Methods. .................................................................................................... 103
3.1 Chemicals and antibodies ............................................................................................. 103
3.2. Cell culture ................................................................................................................... 103
3.3. Selection of stable Bcl-xL- and Bcl-2-overexpressing cell lines ................................. 103
3.4 Transient transfection assay .......................................................................................... 104
3.5. TUNEL assay ............................................................................................................... 104
3.6 Cell counts .................................................................................................................... 104
3.7 Evaluation of mitochondrial membrane potential using a lipophilic cationic dye ....... 105
3.8 Cell viability.................................................................................................................. 105
3.9 Cell fractionation .......................................................................................................... 105
3.10 Protein extraction and Western blotting analysis........................................................ 106
4 Results .............................................................................................................................. 107
4.1 Identification of the EGFP-Bcl-xL and EGFP-Bcl-2 gene expression profiles in GF-1
cells by western blotting and fluorescence microscopy ......................................................... 107
4.2 Overexpression of EGFP-Bcl-xL and EGFP-Bcl-2 prevented GSIV ST kinase-induced
apoptosis and enhanced cell viability. ................................................................................... 107
4.3 Anti-apoptotic proteins Bcl-xL and Bcl-2 blocked GSIV ST kinase-induced MMP loss
................................................................................................................................................ 108
vii
4.4 Bcl-2 and Bcl-xL reduced the extent of the pro-apoptotic protein Bax translocation to
mitochondria and the amount of cytochrome c release. ........................................................ 109
4.5 Downstream events of GSIV-ST kinase protein expression induced caspase-9 and -3
activations in GF-1 cells ........................................................................................................ 109
5 Discussion ........................................................................................................................ 111
6 References: ....................................................................................................................... 134
Chapter 4. ............................................................................................................................. 139
1 Conclusion: ...................................................................................................................... 140
2 Future perspective ............................................................................................................ 140
3 siRNA approach in virus diseases.................................................................................... 141
4 Bcl-2 family overexpression ............................................................................................ 143
5 Antioxidant defense system of host to fight viral invasion.............................................. 144
6 References: ....................................................................................................................... 145
List of peer reviewed Publications...................................................................................... 148
A. Research Papers: ............................................................................................................... 148
B. Posters: .............................................................................................................................. 148
C. Review Papers: .................................................................................................................. 148
D. Book Chapters ................................................................................................................... 149
                                    

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