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研究生:蘇倍儀
研究生(外文):Pei-Yi Su
論文名稱:B型肝炎病毒殼體電荷衡定之研究
論文名稱(外文):Electrostatic homeostasis in hepatitis B virus capsid biology
指導教授:施嘉和
指導教授(外文):Chiaho Shih
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:111
中文關鍵詞:B型肝炎病毒B型肝炎病毒核心蛋白病毒殼體組裝電荷平衡作用空殼病毒Serine 磷酸化
外文關鍵詞:HBVHBcCapsid assemblyElectrostatic interactionsEmpty capsidsSerine phosphorylation
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B型肝炎病毒殼體組裝與穩定性是依據著病毒殼體內包入之核酸(負電)及其核心蛋白上之Arginine-rich Domain(ARD正電)之間的電荷平衡作用。當核心蛋白上ARD正電減少,病毒會傾向包裝較短片段的RNA與DNA;另一方面,當我們逐漸減少核心蛋白上的負電荷(負電荷來自acidic residues與serine磷酸化),這些原本包裝著較短片段的RNA與DNA之病毒可逐漸包裝較長或甚至全長的病毒核酸。類似的電荷平衡作用也可在空殼病毒(empty capsid)上得到驗證。我們將核心蛋白分別表現在三個不同系統:包含昆蟲細胞、大腸桿菌及人類肝臟細胞株,得到的結果皆顯示高程度磷酸化之核心蛋白會造成空殼病毒的產生,而這些空殼病毒在phosphatase去磷酸化後,因為電荷不平衡而導致病毒殼體瓦解;另一方面當我們逐漸增加serine去磷酸化(減少負電),我們能成功地讓空殼病毒逐漸包裝核酸。最後,在提供HBV複製的系統裡,我們發現包入病毒殼體內的病毒RNA與非病毒RNA有著相互補償的現象,此結果可推論除了上述磷酸化機制,細胞本身的RNA也參與著病毒殼體內之電荷平衡作用。我們比較了HBV在非複製與複製系統裡核心蛋白磷酸化之狀況,結果顯示核心蛋白的去磷酸化對於病毒複製機轉是具關聯性的。我們所提出的電荷平衡作用可能對於自然界其他二十面體的病毒殼體組裝是具重要性的。
Capsid assembly and stability of hepatitis B virus (HBV) core protein (HBc) particles depend on balanced electrostatic interactions between encapsidated nucleic acids and an arginine-rich domain (ARD) of HBc in the capsid interior. Arginine-deficient ARD mutants preferentially encapsidated spliced viral RNA and shorter DNA, which can be fully or partially rescued by reducing the negative charges from acidic residues or serine phosphorylation of HBc, dose-dependently. Similarly, empty capsids without RNA encapsidation can be generated by ARD hyper-phosphorylation in insect, bacteria, and human hepatocytes. De-phosphorylation of empty capsids by phosphatase induced capsid disassembly. Empty capsids can convert into RNA-containing capsids by increasing HBc serine de-phosphorylation. In an HBV replicon system, we observed a reciprocal relationship between viral and non-viral RNA encapsidation, suggesting both non-viral RNA and serine-phosphorylation could serve as a charge balance buffer in maintaining electrostatic homeostasis. In addition, by comparing the biochemistry assay results between a replicon and a non-replicon system, we observed a correlation between HBc de-phosphorylation and viral replication. Balanced electrostatic interactions may be important to other icosahedral particles in nature.
Signature Page i
Thesis Approval Form ii
Chinese Abstract iii
English Abstract iv
Table of Contents v
List of Figures vii
List of Tables x
Chapter 1 Introduction -------------------1
1.1 Interests of study --------1
1.2 Life cycle of Hepatitis B virus --------3
1.3 Serine phosphorylation and de-phosphorylation of HBc --------4
1.4 Heterogeneous populations of HBV --------4
1.5 HBV Charge Balance Hypothesis --------5
1.6 Aim of the study --------5
Chapter 2 Results ---9
2.1 The size of encapsidated viral DNA and RNA is determined by the positive charge content at HBc ARD --------9
2.2 The short DNA phenotype of ARD mutants originates from encapsidation of spliced RNAs --------12
2.3 Negatively-charged acidic residues in the full-length HBc context contribute to charge balance --------13
2.4 Serine phosphorylation and de-phosphorylation play a role as a charge balance buffer --------16
2.5 The puzzle of empty capsids in the baculovirus system --------19
2.6 Formation of empty capsids in E. coli via S-to-D mutations --------21
2.7 Serine phosphorylation and HBV RNA packaging in the context without active viral replication in human hepatocytes --------22
2.8 Serine phosphorylation and HBV RNA packaging in the context with active viral replication in human hepatocytes --------24
Chapter 3 Discussion ---27
3.1 Charge Balance Hypothesis --------27
3.2 HBV empty capsids --------28
3.3 The mechanism of HBV RNA encapsidation --------29
3.4 Hierarchy in the position effects of HBc charged residues - --------30
3.5 An electrostatic homeostasis model in the full-length HBc context --------32
3.6 Differences between assays for viral RNA encapsidation and DNA synthesis --------33
3.7 Relationship between major and minor phosphorylations at HBc ARD --------34
3.8 Fixed ratio vs. non-fixed ratio between encapsidated nucleic acids and the net charge of capsid proteins --------35
3.9 Electrostatic interactions in capsid assembly in non-HBV viruses --------36
Chapter 4 Methods ---39
4.1 Plasmids and Cell lines --------39
4.2 Assays for HBV core-associated DNA and RNA --------39
4.3 Native agarose gel and Western blot of core particles --------40
4.4 RT-PCR-sequencing --------40
4.5 PCR of Whole Genome DNA and sequencing --------41
4.6 Baculovirus-expressed HBc capsids in insect cells --------42
4.7 E. coli - expressed HBc capsids --------43
4.8 Preparation of mutant capsids in human hepatoma HuH-7 cells --------43
4.9 Capsid disassembly by micrococcal nuclease or λ-phosphatase treatments --------44
4.10 Phos-tag SDS-PAGE --------45
References 46



List of Figures

Fig 1. A cartoon illustration of the heterogeneity of the genomic contents in naturally occurring HBV particles --------57
Fig 2. A schematic diagram of the domain structure of HBV core protein (HBc) and the amino acid sequences of the C-terminal arginine-rich domain (ARD) --------58
Fig 3. A cartoon illustration of the charge balance hypothesis --------59
Fig 4. Construction of 15 different ARD mutants with different R-to-A substitutions at different positions --------60
Fig 5. Reduction of the positive charge contents of HBc correlated with the reduction of the size of viral genomic DNA by Southern blot analysis --------61
Fig 6. HBc mutants ARD-III and ARD-IV exhibited the most severe defect in HBV RNA encapsidation and DNA synthesis --------63
Fig 7. Reduction of the positive charge contents of HBc correlated with the reduction of the size of viral genomic DNA by Whole Genome DNA PCR --------64
Fig 8. Gradual reduction in the positive charge contents of HBc correlated with the gradual reduction in the size and amount of viral particle-associated HBV RNA --------65
Fig 9. Arginine-deficient HBc mutants preferentially encapsidated spliced viral RNA and DNA --------66
Fig 10. Secreted extracellular viral particles of arginine-deficient HBc mutants preferentially encapsidated spliced viral DNAs --------69
Fig 11. The central concept of Charge Balance Hypothesis --------71
Fig 12. A charge-rebalance approach via reducing acidic residues of HBc --------72
Fig 13. Efficient rescue of defective DNA replication and RNA encapsidation of HBc mutant ARD-III by a charge rebalance approach --------74
Fig 14. Efficient rescue of defective DNA replication and RNA encapsidation of HBc mutant ARD-IV by a charge rebalance approach --------75
Fig 15. Efficient rescue of defective DNA replication and RNA encapsidation of HBc mutant ARD-I+II+III+IV by a charge rebalance approach --------76
Fig 16. Efficient rescue of defective DNA replication and RNA encapsidation of HBc mutant ARD-III+IV by a charge rebalance approach --------78
Fig 17. Replication assay by Southern blot analysis revealed no significant effect on viral DNA synthesis in mutants containing a single rescue mutation in the context of wild type HBc ARD --------80
Fig 18. A charge rebalance approach via reducing serine phosphorylation at HBc ARD --------81
Fig 19. The replication defect of mutant ARD-III can be efficiently rescued by a second mutation S162A --------82
Fig 20. The full-length SS DNA of mutant S162A can be rescued by reducing positive charges from individual HBc ARD --------83
Fig 21. The defective viral replication of HBc mutant ARD-III+IV can be rescued in a dose-dependent manner by reducing negative charges from phosphoserine --------84
Fig 22. Amino acid sequences of HBc ARD of wild type HBV and ten serine-deficient mutants --------86
Fig 23. Empty capsids of wild type HBV generated in the baculovirus system can be converted into RNA-containing capsids by serine de-phosphorylation via S-to-A mutations at HBc ARD --------87
Fig 24. The insect-expressed WT and mutant S7A capsids exhibited distinct morphologies under electron microscopy --------89
Fig 25. Stability of insect-expressed capsids was compared between WT HBc (no encapsidated RNA) and serine-deficient mutants (no ser-phosphorylation) by in vitro treatments with either micrococcal nuclease S7 or phosphatase --------90
Fig 26. Stability of E. coli-expressed capsids was compared between WT HBc (no phosphorylation) and S-to-D HBc ARD mutants (mimicking phosphorylation) by in vitro micrococcal nuclease S7 treatment --------91
Fig 27. The E.coli-expressed WT and mutant S7D capsids exhibited distinct morphologies under electron microscopy --------92
Fig 28. Cartoon illustrations of correlations among different virological and biochemical parameters of HBc VLPs prepared from Baculovirus and E. coli expression systems --------93
Fig 29. Direct assessment of protein phosphorylation by a quantitative Phos-tag gel --------95
Fig 30. Evaluation of the effect of serine phosphorylation at HBc ARD on capsid assembly and RNA packaging in the non-replicon context --------96
Fig 31. Evaluation of the effect of serine phosphorylation at HBc ARD on capsid assembly and RNA packaging in the replicon context --------98
Fig 32. A cartoon summary of three major approaches used here in testing the HBV charge balance hypothesis -------100
Fig 33. HBV RNA encapsidation and DNA synthesis depend on positively-charged residues of HBc ARD -------103
Fig 34. Electrostatic homeostasis of capsid particles plays a critical role throughout the entire HBV life cycle -------105


List of Tables

Table 1. Primer sequences used for mutagenesis at HBc ARD -------109
Table 2. Primer sequences used for E-to-A mutations in the charge rebalance rescue experiments -------109
Table 3. Primer sequences used for S-to-A mutations in the charge rebalance rescue experiments -------110
Table 4. Primer sequences for mutagenesis at the serine residues of HBc ARD -------111
Table 5. Primer sequences for the R-to-K mutations at HBc ARD -------111
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