臺灣博碩士論文加值系統

石斑神經壞死病毒 (grouper nervous necrosis viruses) 可以感染多種經濟魚類的仔魚及稚魚，且致死率最高達 100%，造成養殖漁業極大的損失。本研究以龍膽石斑神經壞死病毒 (DGNNV) 似病毒顆粒 (VLPs) 為材料，分析外殼蛋白之 N 端與雙硫鍵對病毒顆粒組裝及熱穩定性之影響，並利用冷凍電子顯微鏡來解析似病毒顆粒之結構。
當外殼蛋白之 N 端截短 35 個胺基酸後，截短之外殼蛋白失去組裝之能力，而當截短 4、16、25 個胺基酸時，則可組裝為似病毒顆粒。進一步利用點突變的方式，來探討外殼蛋白序列 N 端對病毒顆粒組裝與熱穩定性之影響，結果顯示，雖然所有突變株皆可形成似病毒顆粒，但其相對產量與熱穩定性隨著 Arg 突變數目之增加而降低。當截去 25 個 N 端之外殼蛋白胺基酸時，ΔN25-R29A VLP 與 ΔN25 VLP 在顆粒形成與熱穩定性上，具有相同之特性，因此，Arg29 之突變並不會影響似病毒顆粒的形成及熱穩定性。在單點突變株中，ΔN25-R30A 與 ΔN25-R31A 似病毒顆粒之相對產量與熱穩定性皆低於 ΔN25-R29A 之似病毒顆粒，表示 Arg30 與 Arg 31 對病毒顆粒之形成以及結構穩定性上的維持較重要。當 N 端減少 12 個正電荷胺基酸時 (△N25-R293031A)，似病毒顆粒之產率降低至 1.2 ± 0.9%。
在雙硫鍵之研究中，利用還原劑 β-mercaptoethanol 與點突變來探討雙硫鍵對病毒顆粒組裝之與熱穩定性之影響。在 C187A、C331A 與 C187A/C331A 突變株中皆可形成似病毒顆粒，其大小、外觀與野生型似病毒顆粒相似，而 Cys115 與 Cys201 之點突變卻使外殼蛋白喪失組裝之能力。所有可形成似病毒顆粒之外殼蛋白，皆可拆裝與再組裝，且再組裝率約為 98%。失去雙硫鍵之似病毒顆粒，雖仍可維持其二十面體之結構，但其熱穩定性卻下降了，僅能忍受高溫至 70°C，而失去雙硫鍵之殼粒則無法組裝成病毒顆粒。因此，Cys115-Cys201 分子內雙硫鍵對病毒外殼蛋白次單元之折疊與病毒結構穩定性之維持十分重要。
利用冷凍電子顯微鏡 (Cryo-electron microscopy；Cryo-EM) 技術來觀察未截短 (WT-VLP) 與 N 端截短 25 個氨基酸之似病毒顆粒(ΔN25-VLP)，結果顯示兩者十分相似，直徑約為 380 Å，具有 T = 3 擬等價晶格之外觀。在其結構中可觀察到突出單元 (protrusion domain，介於 ~154-192 Å)、殼層單元 (shell domain，介於 ~112-154 Å) 與殼內之 RNA (＜112 Å)。而每一突出單元 (protrusion domain) 由三個次單元所組成，且依環境之不同，此三個次單元亦有不同的交互作用。N 端 25 個氨基酸的截短並不會影響似病毒顆粒的組裝，且結構與 WT-VLP 相似。藉由 Fourier shell correlation 的方式計算出結構之解析度，得知 WT-VLP 之解析度為 6.5 Å，而 ΔN25-VLP 之解析度為 11.8 Å。

Grouper nervous necrosis viruses belong to the Betanodavirus genus in the Nodaviridae family that is a group of small, non-enveloped icosahedron viruses. More than 30 species of fish are infected by the betanodaviruses, which cause massive mortality in hatchery-reared larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the effects of N-terminus and disulfide bonds of capsid protein on particle formation and thermal stability of grouper nervous necrosis virus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used.

Deletion of 35 residues at the N-terminus completely ruined the VLP assembly. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. Site-directed mutagenesis was used to investigate the effects of N-terminus of capsid protein on particle formation and thermal stability of grouper nervous necrosis virus. Althought all arginine mutants could produce VLPs, the relative amounts and thermal stabilities of arginine-mutated VLPs were decrease. The VLPs from ΔN25-R29A and ΔN25 mutants have similar structural properties on particle formation and thermal stability. Therefore, the effects of Arg29 mutations are negligible. The relative amounts and thermal stabilities of VLPs from ΔN25-R30A and ΔN25-R31A mutants are lower than ΔN25-R29A VLP. When 25 amino acids at N-terminus of DGNNV capsid protein were removed, Arg30 and Arg31 are important for particle formation and particel stability. Although particle could form as 12 positively charged amino acids were lost (△N25-R293031A), the efficiency of particles assembly were decrease to 1.2 ± 0.9% as compare to wild-type VLPs (WT-VLPs).

Site-directed mutagenesis and chemical reducing reagents were used to investigate the roles of disulfide bonds in particle formation and thermal stability of grouper nervous necrosis virus. The homogeneous particles from C187A, C331A and C187A/C331A mutants are indistinguishable from the native virus and WT-VLPs in their sizes and shapes. C115A and C201A mutants could not produce VLPs. The dissociated capsomers from arginine- or cysteine-mutant VLPs all can be reassembled to icosahedrons with efficiencies as high as 100%. When VLP particles are pre-fabricated, the reducing agent cannot disrupt the VLP icosahedron structure. The thiol reduction only caused effects on the disulfide linkages inside the icosahedrons. β-mercaptoethanol-treated WT-VLPs could not tolerate the thermal effects at a temperature higher than 70°C. Once the disulfide linkages in dissociated capsomers were entirely disrupted by β-mercaptoethanol treatment, the resulting capsomers could not reassemble back to icosahedron particles.These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.

In the observation of Cryo-EM, the shapes and sizes of the N-terminus truncated particle (ΔN25-VLP) are indistinct from the full-length particle (WT-VLP). The maximum diameter of DGNNV is approximately 380 Å. Like that of the insect nodaviruses, the surface morphologies of ΔN25-VLP and WT-VLP are consistent with a T = 3 quasi-equivalent lattice. The protrusions (~154 to 192 Å), the inner shell of the capsid (~112 to 154 Å), and the RNA (＜112 Å) were observed in the DGNNV structure. The protrusion domain is consisting of three capsid subunits, and the interactions between these subunits are different. Deletion of 25 residues at the N-terminus did not affect VLPs formation and the structure of ΔN25-VLP is similar to WT-VLPs. Resolutions was calculated by Fourier shell correlation, and the resolution of WT-VLPs and ΔN25-VLPs is 6.5Å and 11.8Å, respectively.

摘要-------------------------------------------------------------------------------------------- I
Abstract---------------------------------------------------------------------------------------- III
目錄-------------------------------------------------------------------------------------------- V
表目錄----------------------------------------------------------------------------------------- X
圖目錄----------------------------------------------------------------------------------------- XI
壹、緒論--------------------------------------------------------------------------------------- 1
貳、文獻回顧--------------------------------------------------------------------------------- 3
一、Betanodavirus 的起源----------------------------------------------------------- 3
二、受到 betanodavirus 感染的魚種及其地理分布---------------------------- 5
三、Betanodavirus 之序列與分類-------------------------------------------------- 8
四、Betanodavirus 的分子生物學-------------------------------------------------- 9
五、感染 betanodavirus 之症狀---------------------------------------------------- 10
六、偵測 betanodavirus 的方式---------------------------------------------------- 10
七、增殖 betanodavirus 之細胞株------------------------------------------------- 13
八、Betanodavirus 的傳染性-------------------------------------------------------- 17
九、發展中之疫苗-------------------------------------------------------------------- 20
十、Betanodavirus 的結構及其穩定性-------------------------------------------- 22
參、外殼蛋白之 N 端影響石斑神經壞死病毒顆粒組裝及熱穩定性------------- 24
一、前言--------------------------------------------------------------------------------- 24
二、材料與方法------------------------------------------------------------------------ 29
1. SSN-1 細胞之繼代--------------------------------------------------------- 29
2. SSN-1 細胞之保存與活化------------------------------------------------ 30
3. 以 SSN-1 細胞增殖 DGNNV------------------------------------------ 31
4. 純化 DGNNV-------------------------------------------------------------- 31
5. 大腸桿菌表現 DGNNV 之外殼蛋白---------------------------------- 32
6. DGNNV 似病毒顆粒之純化 (氯化銫梯度離心法)----------------- 33
7. DGNNV 似病毒顆粒之純化 (蔗糖梯度離心法)-------------------- 35
8. 連續式分光光度計分析-------------------------------------------------- 36
9. 蛋白質定量 (Lowry 法)------------------------------------------------- 37
10. 蛋白質電泳----------------------------------------------------------------- 38
11. 抗VLP 之小鼠血清製備-------------------------------------------------- 39
12. 西方墨點法 (Western blotting) ----------------------------------------- 40
13. 質體的抽取----------------------------------------------------------------- 41
14. DNA 電泳------------------------------------------------------------------- 41
15. 純化膠體中之 DNA------------------------------------------------------ 42
16. 勝任細胞 (competent cell) 的製作------------------------------------- 43
17. 細胞轉型-------------------------------------------------------------------- 43
18. N 端 arginine 突變株之選殖-------------------------------------------- 44
19. 電子顯微鏡負染色樣品之製備與觀察-------------------------------- 44
20. N 端 arginine 突變株似病毒顆粒之純化與分析-------------------- 45
21. N 端 arginine 突變株似病毒顆粒之熱穩定性分析----------------- 45
22. N 端 arginine 突變株似病毒顆粒之拆裝與再組裝----------------- 45
三、結果--------------------------------------------------------------------------------- 47
1. DGNNV 之增殖與純化--------------------------------------------------- 47
2. DGNNV 似病毒顆粒之純化--------------------------------------------- 48
3. DGNNV 與其似病毒顆粒之結構的相似性--------------------------- 51
4. 外殼蛋白之 N 端富含正電荷之胺基酸------------------------------ 52
5. 外殼蛋白 N 端之截短影響似病毒顆粒的形成--------------------- 53
6. 外殼蛋白 N 端之正電荷氨基酸點突變選殖株--------------------- 54
7. N 端 arginine 突變株生產似病毒顆粒之能力分析----------------- 56
8. N 端 arginine 突變株似病毒顆粒之相對產量分析----------------- 61
9. N 端 arginine 突變株似病毒顆粒之熱穩定性分析----------------- 62
10. N 端 arginine 突變株似病毒顆粒之拆裝與再組裝----------------- 67
四、討論--------------------------------------------------------------------------------- 72
1. DGNNV 之增殖------------------------------------------------------------ 72
2. 似病毒顆粒之純化-------------------------------------------------------- 73
3. DGNNV 與其似病毒顆粒之結構十分相近--------------------------- 75
4. 外殼蛋白之 N 端正電荷胺基酸影響似病毒顆粒的形成--------- 76
5. N 端 arginine 突變株似病毒顆粒之熱穩定性----------------------- 80
6. N 端 arginine 突變株似病毒顆粒之拆裝與再組裝----------------- 82
肆、外殼蛋白之雙硫鍵影響石斑神經壞死病毒顆粒組裝及熱穩定性------------ 83
一、前言--------------------------------------------------------------------------------- 83
二、材料與方法------------------------------------------------------------------------ 89
1. 似病毒顆粒中之雙硫鍵分析 (非還原性電泳)----------------------- 89
2. 還原劑 β-mercaptoethanol 對似病毒顆粒之影響------------------- 90
3. Cysteine 突變株之選殖--------------------------------------------------- 91
4. Cysteine 突變株似病毒顆粒之純化與分析--------------------------- 92
5. Cysteine 突變株似病毒顆粒之熱穩定性分析------------------------ 92
6. Cysteine 突變株似病毒顆粒之拆裝與再組裝------------------------ 92
三、結果--------------------------------------------------------------------------------- 93
1. 病毒外殼蛋白中之 cysteine 的保守性分析-------------------------- 93
2. 似病毒顆粒中之雙硫鍵-------------------------------------------------- 94
3. 雙硫鍵對似病毒顆粒的影響-------------------------------------------- 95
4. 外殼蛋白 cysteine 胺基酸點突變選殖株----------------------------- 96
5. Cysteine 突變株生產似病毒顆粒之能力分析------------------------ 98
6. Cysteine 突變株似病毒顆粒之相對產量分析------------------------ 101
7. 雙硫鍵之還原影響野生型似病毒顆粒之熱穩定性----------------- 102
8. Cysteine 突變株似病毒顆粒之熱穩定性分析------------------------ 103
9. 雙硫鍵之還原影響野生型似病毒顆粒之再組裝-------------------- 106
10. Cysteine突變株似病毒顆粒之拆裝與再組裝-------------------------- 107
四、討論--------------------------------------------------------------------------------- 109
1. Betanodavirus 外殼蛋白中 cysteine 之保守性---------------------- 109
2. DGNNV 病毒顆粒中之分子內雙硫鍵--------------------------------- 109
3. DGNNV 外殼蛋白中 Cys115 與 Cys201 形成分子內雙硫鍵--- 110
4. 雙硫鍵之還原會降低似病毒顆粒之熱穩定性----------------------- 112
5. 缺乏雙硫鍵之外殼蛋白次單元無法組裝為似病毒顆粒----------- 114
伍、DGNNV 之冷凍電子顯微結構------------------------------------------------------ 115
一、前言--------------------------------------------------------------------------------- 115
二、材料與方法------------------------------------------------------------------------ 120
1. 電子顯微鏡深染色 (deep stain) 樣品之製備與觀察--------------- 120
2. 電子顯微鏡冷凍樣品 (cryo sample) 樣品之製備與觀察---------- 120
3. 三維立體影像重組之參考影像資料庫-------------------------------- 121
4. 方位角之搜尋與立體結構之重建-------------------------------------- 122
5. 重組立體模型解析度之推算-------------------------------------------- 123
三、結果--------------------------------------------------------------------------------- 125
1. 冷凍電子顯微影像重組-------------------------------------------------- 125
2. DGNNV 似病毒顆粒之冷凍電子顯微結構--------------------------- 126
3. 各個次單元間的交互作用----------------------------------------------- 127
4. 染色法 (negative stain 與 deep stain) 之比較----------------------- 128
四、討論--------------------------------------------------------------------------------- 129
1. DGNNV 似病毒顆粒之冷凍電子顯微影像--------------------------- 129
2. 立體影像重組之指標----------------------------------------------------- 130
3. DGNNV 似病毒顆粒之冷凍電子顯微結構--------------------------- 132
陸、結論--------------------------------------------------------------------------------------- 134
柒、參考文獻--------------------------------------------------------------------------------- 136
捌、圖表-------------------------------------------------------------------------------------- 156
玖、附錄-------------------------------------------------------------------------------------- 268
附錄A、立體影像重組之流程------------------------------------------------------ 268
Fig. A1 The icosahedron structure with 532 point group symmetry.----- 268
Fig. A2 The structure of Nodamura Virus, an alphanodavirus.------------ 269
Fig. A3 Icosahedral particle orientation specification.---------------------- 270
Fig. A4 Any view vector can be referenced to one asymmetric unit
within an icosahedron structure.-----------------------------------------------
271
Fig. A5 Change in projected structure with change in view orientation.- 272
Fig. A6 Schematic representation of generation of PFT data for model
and raw image data.-------------------------------------------------------------
273
Fig. A7 Schematic diagram of EMPFT procedure.------------------------- 274
附錄B、個人著作-------------------------------------------------------------------- 275

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