臺灣博碩士論文加值系統

神經氨酸酶（NA）是流行性感冒病毒膜表上的同源四聚體醣蛋白質，其扮演著在感染過程中讓病毒顆粒解離宿主細胞的角色。由於源自細菌的單體NA就能發揮很好的酵素活性，而已知的流感病毒NA為何得維持四聚體結構尚待探究。本研究自基因庫中收集了2827條NA的氨基酸序列，藉此分析全部的NA亞型，並進一步以反轉錄法證實了28個高度保留的氨基酸序列位點中有21個位點對流感病毒的存活非常重要。神經氨酸酶除了提供重要的酵素活性外，大部份影響病毒存活的高保留位點都座落在NA聚合作用的介面上，由此發現NA的聚合作用對病毒存活是必要的。經研究發現，前述高保留位點在突變後並不影響NA的酵素活性和其在細胞內的定位，但卻影響了NA聚合作用的穩定性。同時，R140A/M也被證實會與負責單碳途徑轉移的絲氨酸羥甲基轉移酶2（SHMT2）作用。然而，在過去十年中以NA為標靶的抗病毒藥物已被廣泛實施，以致A型流感病毒的抗藥株在世界各地迅速擴展和蔓延。因此，進一步了解NA的生物學功能，尤其是NA聚合作用的重要性，以提供更多信息予抗流感病毒學上的發展。

Neuraminidase (NA) is a homotetramer viral surface glycoprotein that is essential for virus releasing during influenza virus infection. It has not been well explored why influenza NA should form a tetramer since the bacterial monomer NA itself already exhibits excellent NA enzymatic activity. A consensus-based scheme has been used to align 2827 NA amino acid sequence collected from GenBank. We concentrated on 28 highly conserved residues among 9 NA subtypes and identified 21 out of the 28 positions as crucial residues for viral survival by reverse genetics. Besides the importance of maintaining NA enzymatic activity, many of these conserved residues were found to locate at the oligomerization interface, suggesting that the oligomerization of NA is essential for viral viability. Further investigation showed that those mutations did not affect NA enzymatic activity and NA cellular localization, but the stability of NA oligomerization. Moreover, a one-carbon pathway transferase -- serine hydroxymethyltransferase 2 (SHMT2) was identified as being associated with R140A/M. While antivirals targeting NA have been widely implemented in the past decade, drug-resistant strains of influenza A viruses have evolved and spread rapidly worldwide. Increased understanding of NA biological functions, in particular, the crucial role of NA oligomerization, could fulfill the knowledge of influenza virus in order to development of antiviral infection.

Table of Contents

指導教授推薦書 i
口試委員會審定書 ii
國家圖書館博碩士論文電子檔案上網授權書 iii
長庚大學博碩士論文著作授權書 iv
Acknowledgments v
中文摘要 vi
Abstract vii
Abbreviations viii
Table of Contents ix
Table of Tables and Figures xi
Table of Appendix xii
CHAPTER I INTRODUCTION 1
1.1 Epidemiology of Influenza A Virus 1
1.2 Human Infection caused by Avian Influenza A Virus 2
1.3 Structure of Influenza A Virus 3
1.4 Influenza A Virus Neuraminidase 4
1.5 Function of Influenza A Virus Neuraminidase 6
1.5.1 Function of Neuraminidase Globular Head 6
1.5.2 Function of Neuraminidase Cytoplasmic Tail 6
1.5.3 Function of Neuraminidase Stalk 7
1.5.4 Other Function of Neuraminidase 7
1.6 Inhibition of Influenza A Virus Neuraminidase .8
1.7 Bacterial Neuraminidase (Sialidase) 10
1.8 Mammalian Neuraminidase (Sialidase) .11
1.9 Objective of the Study 11
CHAPTER II MATERIALS AND METHODS 13
2.1 Sequence analysis and protein structure modeling 13
2.2 Cell lines 13
2.3 Generation of recombinant viruses and plaque assay 14
2.4 Site-directed mutagenesis of NA mutants 14
2.5 RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) 14
2.6 Plaque assay in MDCK cells. 15
2.7 Preparation of FLAG-fused NA protein and western blotting 15
2.8 Analysis of NA enzymatic activity 16
2.9 Confocal microscopy 17
2.10 Computational analysis of protein structure dynamics 18
2.11 Stability of NA dimer/tetramers 18
2.12 Identification of NA associated cellular proteins 19
CHAPTER III RESULTS 21
3.1 In silico analysis of the highly conserved NA residues 21
3.2 Virus rescue of NA mutations on the conserved amino acid residues 22
3.3 Measurement of Ala-substituted NA enzymatic activity 24
3.4 Localization of NA and series mutants in HeLa cells 25
3.5 Distribution of highly conserved NA residues 26
3.6 Structural modeling of Ala substitution on the NA oligomerization interface 27
3.7 Stability of the dimer/tetramer of nonrescued NA mutants 30
3.8 The R140A mutant is specifically associated with SHMT2 32
CHAPTER IV DISCUSSION AND CONCLUSION 35
References 41
Tables 52
Figures and Legends 56
Appendix 73
Curriculum Vitae 92
Table of Tables and Figures
Table 1. The 28 amino acid residues in N1 53
Table 2. Virus rescue of NA mutations on the conserved amino acid residues 54
Table 3. The critical functions of conserved NA residues 55
Fig. 1. A consensus-based approach for the identification of highly conserved amino acid residues in the NA protein 58
Fig. 2. Plaque morphology of recombinant Ala-substituted NA viruses 59
Fig. 3. Measurement of Ala-substituted NA enzymatic activity 60
Fig. 4. Localization of WT NA and nonrescued mutants in HeLa cells62
Fig. 5. Simulation of the NA structure of A/WSN/33 using a SWISS modeling server, with the positions of 28 highly conserved residues labeled 63
Fig. 6. Effects of alanine substitution on the oligomerization interface according to computational analysis 65
Fig. 7. Stability of the oligomerization of nonrescued NA mutants 66
Fig. 8. Immunoprecipitation of cellular proteins using FLAG-NA mutants 68
Fig. 9. The R140A mutant specifically associated with SHMT2 69
Fig. 10. NA activity of R140 series mutants in 293T cells 71
Fig. 11. Key amino acid residues bearing the NA dimer/tetramer
structure 72
Table of Appendix
Appendix Table S1.Dedimerization of mutated NA proteins in Category 3 74
Appendix Table S2.The 28 amino acid residues in N1 updated to Dec, 2011 75
Appendix Fig. S1. A timeline of influenza pandemics and medical advances during the past century 76
Appendix Fig. S2. Illustration of influenza virus particle and viral infection 77
Appendix Fig. S3. Proposed steps in the maturation of influenza virus NA 78
Appendix Fig. S5. Genetic and structural relationships between neuraminidases from different influenza viruses 80
Appendix Fig. S6. Illustration of viral neuraminidase activity 81
Appendix Fig. S7. Cytoplasmic tails and stalk of Neuraminidase managed virion shape and replication rate 82
Appendix Fig. S8. Mechanism of Development of Resistance to Oseltamivir 83
Appendix Fig. S9. Crystal structures of bacterial NA and mechanism of mammalian NA oligomerization 84
Appendix Fig. S10. Map of pFLAG-CMV2-WSN-NA vector 86
Appendix Fig. S11. Map of pcDNA-SHMT2-Mys-His vector 87
Appendix Fig. S12. Schema of a divide-and-conquer, consensus-based analysis for surveying the highly conserved amino acid residues of NA proteins 88
Appendix Fig. S13. Comparison of predicted Ala-substituted NA mutant structure 91

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