Therapeutic in vitro and in vivo approach of influenza virus infection by simultaneous reduction of virus titre and cytokine expression through inhibition of virus-induced {NF-_k63B [NF-kappa-B] and Raf-MEK-ERK activation [Elektronische Ressource] / by Pinto, Ana Ruth Jorge Portugal Machado
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Therapeutic in vitro and in vivo approach of influenza virus infection by simultaneous reduction of virus titre and cytokine expression through inhibition of virus-induced {NF-_k63B [NF-kappa-B] and Raf-MEK-ERK activation [Elektronische Ressource] / by Pinto, Ana Ruth Jorge Portugal Machado

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138 Pages
English

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Therapeutic in vitro and in vivo approach of influenza virus infection by simultaneous reduction of virus titre and cytokine expression through inhibition of virus-induced NF- B and Raf-MEK-ERK activationInaugural Dissertationsubmitted to theFaculty of Medicinein partial fulfilment of the requirementsfor the PhD-Degreeof the Faculties of Veterinary Medicine and Medicineof the Justus Liebig University GiessenbyPinto, Ana Ruth Jorge Portugal MachadoofLisbon, PortugalGiessen 2008From the Institute of Medical VirologyHead: Prof. Dr. Wolfram Gerlichof the Justus Liebig University GiessenFIRST SUPERVISOR AND COMMITTEE MEMBER: Prof. Dr. Stephan PleschkaSECOND SUPERVISOR AND COMMITTEE MEMBER:Prof. Dr. Stephan LudwigCOMMITTEE MEMBERS: Prof. Dr. Heinz-Juergen Thiel (Chairman)Prof. Dr. Juergen LohmeyerDate of Doctoral Defense: 9 December 2008I. Table of ContentsI. Table of contentsI. TABLE OF CONTENTS............................................................................................III. LIST OF FIGURES................................................................................................. VIII. LIST OF TABLES ................................................................................................ VIIV. ABBREVIATIONS .............................................................................................. VIIV. SUMMARY ...........................................................................................................

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Therapeutic in vitro and in vivo approach of influenza virus
infection by simultaneous reduction of virus titre and cytokine
expression through inhibition of virus-induced NF- B and Raf-
MEK-ERK activation
Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfilment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen
by
Pinto, Ana Ruth Jorge Portugal Machado
of
Lisbon, Portugal
Giessen 2008From the Institute of Medical Virology
Head: Prof. Dr. Wolfram Gerlich
of the Justus Liebig University Giessen
FIRST SUPERVISOR AND COMMITTEE MEMBER: Prof. Dr. Stephan Pleschka
SECOND SUPERVISOR AND COMMITTEE MEMBER:Prof. Dr. Stephan Ludwig
COMMITTEE MEMBERS: Prof. Dr. Heinz-Juergen Thiel (Chairman)
Prof. Dr. Juergen Lohmeyer
Date of Doctoral Defense: 9 December 2008I. Table of Contents
I. Table of contents
I. TABLE OF CONTENTS............................................................................................I
II. LIST OF FIGURES................................................................................................. V
III. LIST OF TABLES ................................................................................................ VI
IV. ABBREVIATIONS .............................................................................................. VII
V. SUMMARY ........................................................................................................... XI
VI. ZUSAMMENFASSUNG...................................................................................... XII
1. INTRODUCTION.....................................................................................................1
1.1. Influenza viruses ................................................................................................1
1.1.1. Different types of influenza viruses ................................................................1
1.1.2. Influenza A virus ............................................................................................2
1.1.2.1. Morphology and genome structure of influenza A virus...............2
1.1.2.2. Propagation and genome replication of influenza A virus............5
1.1.3. Antigenic variation of influenza virus infection .............................................10
1.1.4. Avian influenza viruses ..........................................11
1.1.4.1. History ...................................................................................................11
1.1.4.2. Current situation (epidemics and pandemics) ..............12
1.1.5. Clinical symptoms of influenza virus infection..............................................13
1.2. Mechanisms of intracellular signal transduction and influenza A viruses .15
1.2.1. The MAPK pathway (Raf/MEK/ERK signalling cascade).............................16
1.2.1.2. Role of Raf/MEK/ERK signalling cascade in influenza A virus infection 19
1.2.2. The NF- B pathway.....................................................................................21
1.2.2.1. Role of NF- B signalling cascade in influenza A virus infection ............24
1.3. Immune response and cytokine interplay......................................................25
1.4 Aims....................................................................................................................32
2. MATERIALS AND METHODS..............................................................................34
2.1. Materials............................................................................................................34
2.1.1. Instruments..........34
2.1.2. Reagents and general materials ..................................................................35
2.1.3. Monoclonal and polyclonal antibodies.......37
2.1.4. Materials for cell culture ...............................................................................37
2.1.5. Materials for mice experiments ..................38
2.1.6. Kits...............................................................................................................38
2.1.7. Virus strains and cell lines ...............................................................38
2.1.8. Inhibitors and solvent.................................38
II. Table of Contents
2.1.9. Media...........................................................................................................39
2.1.10. Buffers and solutions ..................40
2.1.11. Gels and other media.................................................................................42
2.2. Methods.............................................................................................................44
2.2.1. Working with cell cultures ............................................................................44
2.2.1.1. Maintenance of cell culture....................................................................44
2.2.1.2. Storage of cell cultures........................44
2.2.1.3. Infection of cells........................................................................45
2.2.2. Preparation of cell lysates for Western blot analysis...........45
2.2.3. Cell viability (cytotoxicity) analysis ...............................................................46
2.2.3.1. MTT-assay ...................................................................46
2.2.3.2. WST-1-assay.......................................47
2.2.3.3. Trypan Blue dye exclusion ...........................................47
2.2.4. Raising virus stocks ............................................................47
2.2.5. Analysis of infectious virus titres by immunohistochemistry.........................48
2.2.6. Haemagglutination (HA) Assay....................................................................49
2.2.6.1. Preparation of red blood cells (RBCs) from chicken blood.......49
2.2.6.2. HA assay...............................................................................................49
2.2.6.3. HI assay .....................50
2.2.7. Confocal Laser Scanning Microscopy and Immunofluorescence Assay (IFA)
...............................................................................................................................50
2.2.8. Western blotting (Semi-dry)..........51
2.2.8.1. Measurement of relative protein concentration (Bio-Rad protein assay)
...........................................................................................................................51
2.2.8.2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
...........................................................................................................................51
2.2.8.3. Transfer to membrane in a "Semi-dry" electroblotter.............................52
2.2.8.4. Immunodetection of proteins .................................................................52
2.2.8.5. Enhanced Chemiluminescence (ECL) reaction.....................................53
2.2.8.6. Quantification of protein bands.....................................53
3. RESULTS..............................................................................................................57
3.1. Viability of A549 cells upon treatment with specific inhibitors ....................57
3.2. Virus infection induces the NF-B signal cascade in A549 cells and Bay 11-
7082 can inhibit this activation as well as decrease virus titres.........................58
3.3. Virus infection induces the Raf/MEK/ERK signal cascade in A549 cells and
U0126 can inhibit this activation as well as decrease virus titres ......................61
3.4. Bay 11-7082 and U0126 can decrease influenza A virus-induced cytokine
production in A549 cells.........................................................................................64
3.5. FPV and PR8-induced nuclear RNP export is efficiently blocked by Bay 11-
7082 and U0126 in A549 cells.................................................................................66
Primary mice alveolar epithelial cells (AECs).......................................................69
III. Table of Contents
3.6. Viability of mice primary alveolar epithelial cells upon treatment with
specific inhibitors....................................................................................................69
3.7. Viability of mice primary alveolar epithelial cells upon treatment with
specific inhibitors during the course of infection ................................................70
3.8. Both Bay 11-7082 nd U0126 can decrease virus titres in mice primary AECs
..................................................................................................................................72
3.9. Bay 11-7082 and U0126 can decrease influenza A virus-induced cytokine
production in mice primary AECs..........................................................................74
3.10. FPV and PR8 induced nuclear RNP export is efficiently blocked by Bay 11-
7082 and U0126 in AECs.........................................................................................76
3.11. Cell viability in A549 cells with combination treatment (Bay and U0126). 79
3.12. Virus titres treated with combination treatment (Bay and U0126). ............80
3.13. Cell viability in AECs with combination treatment (Bay and U0126). ........81
3.14. C57BL/6 mice..................................................................................................82
3.14.1 Bay 11-7082 and U0126 can decrease virus titres in in C57BL/6 mice ......83
3.14.2 Bay 11-7082 and U0126 can decrease virus induced cytokines in C57BL/6
mice .......................................................................................................................84
4. DISCUSSION ........................................................................................................90
4.1. Bay 11-7082 can inhibit virus-induced NF-B activation in A549................91
4.2. Bay 11-7082 can inhibit virus titres in in vitro cell cultures as well as in vivo
..................................................................................................................................92
4.3. U0126 can inhibit virus-induced the Raf/MEK/ERK activity in A549 cells...94
4.4. U0126 can inhibit virus titres in in vitro cell cultures as well as in vivo......94
4.5. Bay 11-7082 and U0126 can decrease influenza A virus-induced cytokine
production in in vitro cell cultures as well as in vivo...........................................95
4.6. Both FPV and PR8-induced nuclear RNP export is efficiently blocked by
Bay 11-7082 and U0126 in A549 and AECs. ........................................................100
4.7. Combination treatment does not enhance individual inhibitor-induced
decrease in virus titres .........................................................................................102
5. CONCLUSIONS..................................................................................................103
6. REFERENCES....................................................................................................106
7. DECLARATION ..................................................................................................117
IIII. Table of Contents
8. CURRICULUM VITAE.........................................................................................118
9. ACKNOWLEDGEMENTS...................................................................................122
IVII. List of Figures
II. List of Figures
FIGURE 1.1: THE RESERVOIR OF INFLUENZA A VIRUSES ................................................................... 11.2.A: CARTOON OF THE INFLUENZA VIRION............................................................... 3
FIGURE 1.2.B: THE INFLUENZA VIRUS RNP STRUCTURE..................................................................... 51.3: THE REPLICATION CYCLE OF INFLUENZA VIRUS......................................................... 7
FIGURE 1.4: INFLUENZA A VIRUS-ACTIVATED SIGNALLING PATHWAYS ................................... 151.5: OVERVIEW OF RAS/RAF/MEK/ERK PATHWAY. ................................................... 18
FIGURE 1.6: LATE ACTIVATION OF RAF/MEK/ERK PATHWAY IN INFLUENZA A VIRUS
INFECTION............................................................................................................................................... 20
FIGURE 1.7: OVERVIEW OF NF- B PATHWAY........................................................................................ 231.8: CYTOKINE INTERPLAY. ........................................................................................................ 28
FIGURE 1.9: THE TWO CELLULAR PATHWAYS TARGETED IN THIS STUDY (NF- B) AND
(RAF/MEK/ERK). ..................................................................................................................................... 32
FIGURE 3.1: VIABILITY TEST ON A549 CELLS........................................................................................ 583.2.1: VIRUS-INDUCED NF-B ACTIVITY CAN BE REDUCED BY BAY ................... 59
FIGURE 3.2.2: VIRUS TITRES CAN BE REDUCED BY BAY.................................................................... 603.3.1.A: FPV-INDUCED ERK ACTIVITY CAN BE REDUCE BY U0126. ....................... 61
FIGURE 3.3.1.B: PR8-ITY CAN BE REDUCE BY U0126. .................................. 623.3.2: VIRUS TITRES ARE DECREASED BY U0126 TREATMENT. ....................................... 63
FIGURE 3.4.1: VIRUS-INDUCED CYTOKINE RELEASE IS DECREASE UPON INHIBITOR
TREATMENT............................................................................................................................................ 64
FIGURE REASE U
TREATMENT............................................................................................................................................ 65
FIGURE 3.4.2: IFN  INDUCTION IN A549 CELLS...................................................................................... 663.5.1: VRNP NUCLEAR EXPORT IN A549 CELLS.................................................. 67
FIGURE 3.5.2: VRNP NU..................................................................... 683.6: VIABILITY TEST ON MICE PRIMARY ALVEOLAR EPITHELIAL CELLS................. 70
FIGURE 3.7: VIABILON MICE PRIMARY ALVEOLAR EPITHELIAL CELLS DURING
INFECTION............................................................................................................................................... 71
FIGURE ITY TEST .............................................................................................................................................. 72
FIGURE 3.8: VIRUS TITRES IN MICE PRIMARY ALVEOLAR EPITHELIAL CELLS UPON
TREATMENT............................................................................................................................................ 73
FIGURE 3.9.1 (A AND B): CYTOKINE INDUCTION IN PRIMARY ALVEOLAR EPITHELIAL
CELLS. ....................................................................................................................................................... 74
FIGURE 3.9.1 (C AND D): CYTOKINE INDUCTION IN PRIMARY ALVEOLAR EPI
CELLS. ....................................................................................................................................................... 75
FIGURE 3.9.2: IFN  INDUCTION IN PRIMARY ALVEOLAR EPITHELIAL CELLS. ............... 763.10.1: VRNP NUCLEAR EXPORT IN MICE PRIMARY ALVEOLAR EPITHELIAL CELLS.
..................................................................................................................................................................... 77
FIGURE 3.10.2: VRNP NUCLEARICE
..................................................................................................................................................................... 78
FIGURE 3.11: VIABILITY OF A549 CELLS WITH COMBINATION TREATMENT (BAY + U0126). 793.12: VIRUS TITRES WITH COMBINED INHIBITORS ON A549 CELLS.............................. 80
FIGURE 3.13: MICE PRIMARY ALVEOLAR EPITHELIAL CELLS VIABILITY TEST (WST ASSAY)
WITH COMBINATION TREATMENT................................................................................................. 81
FIGURE 3.14: VIRUS TITRES IN MICE TREATED WITH INHIBITORS............................................... 843.15: TNF-  RESULTS IN MICE TREATED WITH INHIBITORS............................................ 85
FIGURE 3.16: IFN  RESULTS IN MICE TREATED WITH INHIBITORS. ............................................. 863.17: KC (A) AND MCP-1 (B) RESULTS IN MICE TREATED WITH INHIBITORS.............. 87
FIGURE 3.18: IL-6 (C) AND RANTES (D) RESULTS IN MICE TREATED WITH INHIBITORS. ....... 88
VIII. List of Tables
III. List of Tables
TABLE 1.1.: INFLUENZA A VIRUS PROTEINS AND FUNCTIONS (STRAIN A/PR/8/34, H1N1)....... 3
TABLE 1.2: LIST OF SOME IMPORTANT CYTOKINES AND CHEMOKINES IN INFLUENZA VIRUS
INFECTION.. ................................................................................................................................ 31
TABLE 3.1.: SUMMARY OF REDUCTION OF CYTOKINE/CHEMOKINE BY THE SPECIFIC
................................................................................................................................. 89INHIBITORS
VIIV. List of Abbreviations
IV. Abbreviations
aa amino acids
A549 human alveolar epithelial cell line
AEC mice primary alveolar epithelial cell
AI avian influenza
AIV avian influenza virus
AMs alveolar macrophages
APCs antigen-presenting cells
APR acute phase response
APS ammonium persulfate
ARDS acute respiratory distress syndrome
BSA bovine serum albumin
°C degree celcius
cm centimetre
CTL cytotoxic T lymphocytes
delNS1 A/PR/8/34 with a deleted NS segment
ddH O deionized distilled water2
DMEM Dulbecco's Modified Eagle's medium
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
dNTP deoxynucleoside triphosphate
DTT dithiothreitol
EBV Epstein-Barr virus
ECL enhanced chemoluminiscence
EDTA ethylenediamine tetraacetic acid
eIF2 eukaryotic translation initiation factor 2
ERK extracellular signal regulated protein kinase
et al. et alii (and others)
FasL Fas ligand
FCS fetal calf serum
FFU foci forming unit(s)
FPV fowl plague virus
g gram
VIIIV. List of Abbreviations
h hour(s)
HA hemagglutinin
HEPES N-2-hydroxyethylpiperazine
HIV human immunodeficiency virus
HPAIV highly pathogenic avian influenza virus
HTLV human
IAV influenza A virus
IBV influenza B virus
IFN interferon
I B    inhibitor of NF- B alpha
IKK I B kinase
IP-10 IFN-  inducible protein-10
IRF3 IFN regulatory factor 3
IL- 6/8 interleukin 6/8
JNK c-Jun NH -terminal kinases2
LPAIV lowly pathogenic avian influenza virus
M molar
mAbs monoclonal antibodies
M1 matrixprotein
M2 ion channel protein
MAPK mitogen-activated protein kinase
MAPKK MAPK kinase
MAPKKK MAPKK kinase
MEK MAPK/ERK activated kinase
MEKK MAPK/ERK activated kinase Kinase
mg milligram
MHC major histocompatibility complex
MIG monokine-induced by IFN- 
min minute(s)
MIP macrophage inflammatory protein
ml milliliter
mM millimolar
moi multiplicity of infection
VIII