Title Page

Contents

List of Abbreviations 13

Abstract 16

Literature Revies 18

1. Avian viruses 19

1.1. Influenza virus 19

1.2. Newcastle disease virus 25

2. Host defense against virus infection 30

2.1. Pattern recognition receptor (PRR) 30

2.2. Interferon system 36

2.3. Interferon-induced antiviral effectors 39

3. Interferon induced Mx GTPase family 44

3.1. Family members 44

3.2. Dynamin-like structure of Mx protein 47

PART I. Over-expression of Chicken Mx Protein in Chicken Embryonic Fibroblast Cell Line and Primary Tracheal Epithelial Cells Inhibits Avian Viruses Replication and Progeny Release 50

1. Introduction 51

2. Materials and Methods 54

2.1. Cells 54

2.2. Viruses 55

2.3. Construction of plasmids 55

2.4. Immunofluorescence assay (IFA) 56

2.5. Western blot analysis 57

2.6. cDNA synthesis 59

2.7. Conventional RT- PCR 59

2.8. Production of chMx specific antibody 60

2.9. Establishment of chMx or N631S over-expressing cell lines and confirmation 60

2.10. Virus infection 61

2.11. Virus progeny titration assay 61

2.12. Flow cytometry analysis 62

2.13. Real-time RT-PCR 63

2.14. Statistical analysis 63

3. Results 66

3.1. Analysis of chMx functional domains 66

3.2. Construction and expression of chMx and mutant N631S protein 66

3.3. chMx-specific poly-clonal antibody production 71

3.4. Establishment of chMx or N631S over-expressing cell lines and confirmation 73

3.5. Inhibition of influenza virus and NDV replication in chMx or N631S over-expressing cell lines 75

3.6. Inhibition of influenza virus and NDV replication in chMx or N631S transiently over-expressing chicken tracheal primary cell culture 80

3.7. Down-regulation of viral mRNA transcription by chMx over-expression 81

3.8. chMx dose-dependent inhibition of virus replication 87

3.9. Over-expression of Mx inhibited virus progeny release 90

4. Discussion 92

PART II. Molecular Approaches for Characterization of ChMx Putative 2ndORF Function 97

1. Introduction 98

2. Materials and Methods 100

2.1. Cells and viruses 100

2.2. Construction of plasmids 100

2.3. Immunofluorescence assay (IFA) 101

2.4. Co-immunoprecipitation (co-IP) 102

2.5. Western blot analysis 104

2.6. Virus infection 104

2.7. cDNA synthesis 105

2.8. Conventional RT-PCR 105

2.9. Real-time RT-PCR 106

2.10. Statistical analysis 106

3. Results 107

3.1. Mx-specific antibody detects two Mx bands 107

3.2. rNDV-GFP transduction partial induces nuclear localization of chMx distribution 107

3.3. Predicted nuclear export signal (NES) in chMx 108

3.4. Characterization of chMx first 47amino acid 108

3.5. Different expression pattern of chMx 2ndORF compared with wild type chMx 114

3.6. 2ndORF oligomerizes with chMx 115

3.7. 2ndORF inhibits influenza virus replication 119

3.8. 2ndORF efficiently inhibits virus replication 119

3.9. M48A construction and expression 121

3.10. ChMx 2ndORF may interact with influenza virus NP 124

4. Discussion 127

PART III. Establishment of air-liquid interface transwell system for trachea epithelia mimic model 130

1. Introduction 131

2. Materials and Methods 132

2.1. Cells 132

2.2. Organo-culture 132

2.3. Scanning electron microscopy (SEM) 132

2.4. Viruses 133

2.5. cDNA synthesis 133

2.6. Real-time RT-PCR 133

3. Results 134

3.1. Confirmation of chicken primary trachea epithelial cells (TECs) 134

3.2. Establishment of air-liquid interface trachea mimicking model system 134

3.3. Application of trachea mimic system in influenza virus infection 135

4. Discussion 140

References 141

국문초록 165

Table 1. Lists of PRRs and viral/non-viral PAMPs 29

Table 2. Primers used in PART I 58

Table 3. Real-time RT-PCR primers used in PART I 65

Table 4. Primer list used in PART II 103

Figure 1. Sturcture of influenza A virus. Figure represents a matured single virion composition which is constituted as polymerase complex (PB2, PB1, PA), nucleocapsid protein (NP), glycoproteins (HA, NA, ion-channel protein M2), structural protein (matrix M1, nuclear export protein NEP) and nonstructural protein (NS1). 22

Figure 2. Single cell reproductive cycle of influenza A virus. 24

Figure 3. Newcastle disease virus genome structure. 26

Figure 4. Newcastle disease virus (NDV) life cycle. 28

Figure 5. Toll like receptors (TLRs) and RIG-I like helicases (RLHs) signaling. 34

Figure 6. Interferon receptor signaling. 38

Figure 7. Phylogenetic tree of Mx proteins 46

Figure 8. Structure and domain composition of Mx GTPases. The GTP-binding domain (light blue) contains the tripartite GTP-binding element and the self-assembly domain (SAD). The C-terminal effector domain contains the central interactive domain (CID) and a leucine zipper region (LZ), also called GTPase effector domain (GED) in the case of dynamin. In addition,... 49

Figure 9. Construction of plasmid wild-type chMx and N631S containing single amino acid mutation at 631 residue, and analysis of functional domain. (A) To amplify Mx gene, CEF (Chicken embryonic fibroblast) cell line was treated with LPS (Lipopolysaccharide) and poly I:C for 16hrs and total RNA was extracted. Random hexamer was used for cDNA synthesis,... 69

Figure 10. Expression of chMx and mutant N631S protein. RD cells were transiently transfected with pcDNA-chMx(A) or pcDNA-N631S plasmid DNA(B), and protein expression was analyzed by IFA and Western blot. Expressed proteins are detected by anti-6X His antibody, and visualized by FITC(fluorescein isothiocyanate)-labeled secondary antibody under confocal... 70

Figure 11. ChMx-specific polyclonal antibody production. Three chMx B cell epitopes were predicted along with the amino acid sequence information (A). Synthesized chMx B cell epitope peptide-specific antibodies were raised in rabbit and examined biological activity by Western blot analaysis. ChMx-specific antibody (Ab) #2 showed the highest avidity against transiently... 72

Figure 12. Establishment of chMx and N631S over-expressing cell lines and confirmation. Established over-expressing cell lines were confirmed for chMx (A) and N631S (B) expression by IFA. Transcripts and protein expression were confirmed by RT-PCR and Western blot analysis, repectively (C). Samples were normalized by β-actin in Western blot (WB) and... 74

Figure 13. Avian influenza and Newcastle diease virus (NDV) infection in chMx and N631S over-expressing cell lines. (A) The chMx, N631S over-expressing CEF cell lines and normal CEF cell line were infected with 0.1 m.o.i for 40 min and incubated for 24 or 48hrs. (B) Each viral proteins was detected with H1N1, H9N2 or NDV infected chicken hyperimmunesera as a... 79

Figure 14. AIVs and NDV infection in transiently chMx or N631S plasmid transfected primary chicken tracheal epithelial (CTE) cells. (A) Isolated primary CTE cells were confirmed by detecting representative epithelial cell marker such as cytochrome P-450 2C45, Keratin 14 and Retinoic acid responder by conventional RT-PCR. (B) Cells were grown on 6- well plate and... 85

Figure 15. ChMx or N631S over-expression in primary chicken tracheal epithelial (CTE) cells lowered viral mRNA expression level. ChMx or N631S transiently transfected primary CTE cells were infected with H1N1 (A) or H9N2 (B) at 0.1 m.o.i. At 24hrs p.i., total cellular RNA was extracted and and subjected for cDNA synthesis which is used for real-time RT-PCR with... 86

Figure 16. ChMx-dependent inhibition of viral replication. Primary CTE cells were transiently transfected with increasing amount of chMx by 0, 1 or 2ug. At 48hrs p.i., cells were infected for 24hrs with H1N1 or H9N2 at 0.1 m.o.i. Harvested cells were lyzed and analyzed by Western blot analysis (A). Results were further analyzed by Quantitity OneR to calculate relative...(이미지참조) 89

Figure 17. Infectivity assay of viral progeny produced from H1N1 infected cell culture. Transiently chMx or N631S plasmid transfected primary CTE cells were infected with H1N1 at 0.1 m.o.i. Culture supernatants were harvested at 24 and 48 hrs p.i. and anti-viral effect of over-expressed chMx proteins were measured by end point dilution assay... 91

Figure 18. Two chMx bands which are detected by Mx-specific or 6X His tag-specific antibody. chMx-specific antibody detects B cell epitope of chMx peptide 68 to 83, both upper and lower band were detected. 82.8kDa molecular mass of chMx or N631S bands were detected by this antibody together with approximately 5 kDa smaller band. Also this samller band was detected... 110

Figure 19. Partially nuclear localized chMx distribution. rNDV-GFP was transduced into the chMx over-expressing cell line grown on 12-well plate. 24hrs later, cells were fixed, and chMx-specific antibody was treated at 1:1000 as primary antibody. Samples were visualized by confocal microscope. 111

Figure 20. ChMx has putative nuclear exporting signal (NES) which influences sub-cellular localization. Full length chMx amino acid sequences were subjected to NET-NES 1.1 prediction, NES signal was analyzed between two start codon. 112

Figure 21. Characterization of chMx first 47 amino acids. Influenza virus H1N1 NP coding plasmid, chMx first 47 amino acids and chMx first 47 amino acids fused with NP were constructed (A). Expression of both NP and chMx47aa-NP construct was identified in a single cell (B) 113

Figure 22. Construction and expression of chMx 2ndORF. 2ndORF is constructed by truncation of amino acids from 2nd to 48th, second start codon(A). Single cell expression was confirmed by IFA, and 2ndORF shows different expression pattern compared with any other Mx constructs, chMx and N631S (B). Western blot analysis revealed that lower size band induced by chMx or...(이미지참조) 117

Figure 23. 2ndORF forms oligomer with chMx. Four ug of FLAG-Mx was co-transfected with 4ug of each pcDNA-chMx or pcDNA-2ndORF. 48hrs later, cells were lyzed and harvested SPNTs were subjected to co-IP using anti-FLAG antibody. Protein G agarose was used for capture formed complex, samples were analyzed by Western blot analysis. 118

Figure 24. Infection study using chicken primary TECs. Primary cells were grown onto 6-well plate, and further transfected with each 2ug of DNA. 48hrs later, cells were infected with H1N1 or H9N2 at 0.1 m.o.i. Cells were harvested at 24 or 48hrs post infection, SPNTs from lyzed cells were subjected to SDS-PAGE followed by Western blot analysis with virus infected... 120

Figure 25. 2ndORF efficiently inhibits virus replication. H1N1 infection at 0.1 m.o.i. to primary cells which is previously transfected by three constructs was analyzed by wWstern blot analysis (A). Band density in Western blot analysis was further analyzed by Quantity One (B). Real-time RT-PCR was conducted to demonstrate relative expression of each constructs together with... 122

Figure 26. M48A construction and expression. M48A was constructed by single amino acid substitution at M48, and it was expressed in spot-shaped pattern with a single upper band. 123

Figure 27. chMx 2ndORF may interact with influenza virus NP. One ug of each chMx, M48A or 2ndORF was co-transfected with one ug of NP to HeLa cell and visualized by chMx-specific antibody or NP detectable anti 6X His antibody (A). Co-transfection of chMx, M48A or 2ndORF with NP to 293T cells was subjected to co-IP, further analyzed by SDS-PAGE... 125

Figure 28. chMx did not degrade influenza virus NP directly. Fixed amount of NP plasmid was co-transfected with gradually increased amount of chMx to 293T cell. chMx and NP were detected using chMx-specific or NP detectable anti 6X His antibody. 126

Figure 29. Confirmation of chicken primary trachea epithelial cells (TECs). Isolated cells were maintained in growth media, and epithelial cell morphology was observed (A). Tracheal epithelial cells from 10-day old embryonated chicken egg were observed under scanning electron microscope (SEM). Scale bar indicates 3 μm distance (B). Chicken primary TECs from... 136

Figure 30. Establishment of air-liquid interface trachea mimic system. Schematic representation of the system (A), and TECs were harvested and applied to transwell system (B). 137

Figure 31. Organo-culture model system on transwell. Schematic representation of this system (A), and the tracheas are directly applied to membrane transwell and maintained by supplying growth media (B). 138

Figure 32. Application of trachea mimic system in influenza virus infection. Real-time RT-PCR was conducted with total RNA extracted from monolayered TECs or trachea tissue (A, B). 139

인터페론에 의해 발현되는 유전자 중 하나로, Mx 단백질은 1 형 인터페론에 의해 세밀하게 조절된다. Mx 는 여타 다른 dynamin 계열의 단백질처럼 GTP 가 결합하는 도매인을 가지며 비교적 높은 수준으로 보존되는 부분이 단백질의 N 말단 부위에 존재한다. 현재까지 Mx 단백질이 단일 세포 내에서 작용하는 매커니즘은 잘 규명되어 있지 않다. 몇몇 보고에 따르면 세포질에서 발현되는 인간 MxA 나 핵에서 발현되는 쥐 Mx1 의 경우 인플루엔자 바이러스의 감염에 대해 바이러스 복합체의 구성요소인 PB2 나 NP 와의 결합을 통해 복제를 저해함으로써 저항성을 확보하는 것으로 알려져있다. 닭의 Mx 와 그의 돌연변이 형인 N631S 가 실험에 사용되기 위하여 포유류 발현 벡터인 pcDNA3 에 클로닝 되었다. 닭 Mx 과발현 세포주가 확립되어 바이러스 감염 실험에 활용되었다. 10 일령의 수정란에서 유래한 기도 상피 세포의 일차배양을 통해 자연 상태와 근접한 환경을 조성한 감염 실험도 진행되었다. H1N1, H9N2 인플루엔자 바이러스와 Newcastle disease virus (NDV) 를 닭 Mx 안정 세포주에 감염시켜 Mx 의 발현에 따른 저항성을 알아보았다. 조류 바이러스의 세포 내 복제는 닭 Mx 가 농도 의존적으로 과발현 되었을 때 전사 및 번역 단계에서 각각 감소하는 것이 확인되었다. 또한 GFP 단백질을 발현하는 재조합 NDV 를 이용한 감염 및 Flow cytometry 분석법을 통해 정량적인 저해 양상을 확인할 수 있었다. 반복적으로 수행된 선행 실험을 통해 닭 Mx 에 또 다른 형태의 Mx 단백질이 존재 함을 확인할 수 있었으며, 아미노산 수준의 분석을 통해서 닭 Mx 의 초반 47 의 아미노산에서 핵 외로 단백질의 이동을 돕는 신호단백질이 존재함을 추정할 수 있었다. 이의 정확한 분석을 위하여 두번째 번역 시작 codon 인 48 번째 메치오닌을 알라닌으로 치환한 M48A 와 초반 47 개 아미노산을 배제한 2ndORF vector 가 구축되었으며, 닭 Mx 의 초반 47 개 아미노산과 인플루엔자 바이러스의 NP 단백질이 결합된 벡터를 구축하여 닭 Mx 의 2ndORF 가 인플루엔자 바이러스의 복제 저해 기작에 미치는 영향을 분석하였다. 또한 닭 기도와 유사한 시스템을 구축하여 자연 상태에 근접한 상황에서 각종 호흡기도에 감염되는 바이러스의 연구를 돕고자 하였다. 이 시스템은 공기와 직접 맞대고 있는 기도의 상피세포를 분리해내어 자연상태에 근접하게 공기중에 노출시키는 배양기법으로서 바이러스의 감염시 나타나는 현상의 분석에 중요하게 사용될 수 있을 것으로 기대한다. 본 연구에서는 분리된 닭 기도의 조직배양도 수행되었으며 정상적으로 감염 실험 등에 적용될 수 있음을 확인하였다.