Functional analysis of cytosolic sensors of viral nucleic acids and their role for innate antiviral immune defense [Elektronische Ressource] / Katharina Eisenächer

Katharina Eisenächer

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 32
• Langue: Deutsch
• Poids de l'ouvrage: 26 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Humanbiolgie
Functional analysis of cytosolic sensors of viral
nucleic acids and their role for innate antiviral
immune defense
Katharina Eisenächer
Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.
Vorsitzende: Univ.-Prof. Dr. H. Daniel
Prüfer der Dissertation: 1. Univ.-Prof. Dr. M. Schemann
2. Priv.-Doz. Dr. A. Krug
3. Prof. Dr. Adolfo García-Sastre, Ph.D.
(Mount Sinai School of Medicine, New
York, USA)
Die Dissertation wurde am 04.10.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 10.02.2011 angenommen.

















Antoine de Saint-Exupéry ★ Le Petit Prince



TABLE OF CONTENTS
TABLE OF CONTENTS
Table of Contents ............................................................................................................ I
Index of Figures............ VI
Index of Tables .......................................................................................................... VIII
Abbreviations................ IX
1 Introduction............................................................................................................1
1.1 The innate immune system protects from invading pathogens.........................1
1.2 Pattern recognition receptors...............2
1.3 RIG-I-like receptors are viral RNA sensors........................................................2
1.3.1 Identification of RIG-I and its function.....................3
1.3.2 MDA5........................................................................................................5
1.3.3 Downstream signaling is mediated by the adaptor protein IPS-1 .............6
1.3.4 LGP2..........7
1.3.5 Viral recognition by RIG-I-like receptors .................................................8
1.3.6 RLRs are accurate sensors of virus invasion – discrimination between .....
self and non-self ......................................................11
1.4 RLR-mediated signal transduction....................................................................13
1.5 Regulation of RLR signaling by cellular proteins............17
1.5.1 Role of ubiquitination for activation and degradation of RIG-I.............18
1.5.2 RIG-I splice variant functions as off-switch regulator of its own
pathway....................................................................................................20
1.5.3 Regulation of MDA5...............20
1.5.4 Regulation of RLR signaling at the level of IPS-1..21
1.5.5 Other mechanisms involved in the regulation of RLR signaling ............22
1.6 Interaction between RLRs and viral proteins...................................................24
1.6.1 Viral escape from recognition by the RLR system .................................24
1.6.2 Disruption of the interaction of signaling molecules ..............................25
1.6.3 Cleavage or degradation of signaling molecules.....27
1.7 Involvement of RLRs in autoimmunity .............................................................29
2 Aims of the study .................................................................33
3 Material & Methods............................................................35
I TABLE OF CONTENTS
3.1 Material ................................................................................................................35
3.1.1 Primers.....35
3.1.2 Plasmids...................................................................................................35
3.1.2.1 Commercially available and published plasmids ...............................35
3.1.2.2 Plasmids constructed in this project...................................................36
3.1.3 Antibodies and protein standard for IFN-β enzyme-linked ........................
immunosorbent assay ..............................................37
3.1.4 Antibodies................................................................38
3.1.5 Cell lines, virus strains and bacterial strains...........38
3.1.5.1 Cell lines.............................................................38
3.1.5.2 Virus strains........................................................39
3.1.5.3 Bacterial Strains.................................................39
3.1.6 Buffers, solutions and media ...................................40
3.1.6.1 Media for cell culture.........40
3.1.6.2 Solutions for cell culture applications................................................42
3.1.6.3 Buffers for cytokine ELISA .................................................................42
3.1.6.4 Media for bacterial applications........................43
3.1.6.5 Buffers and solutions for protein biochemistry...44
3.1.6.6 Buffers for molecular biology.............................................................47
3.2 Methods ................................................................................48
3.2.1 Mice.........................................48
3.2.2 Tissue culture...........................................................................................48
3.2.2.1 Cell lines.............................48
3.2.2.2 Cryopreservation of cells....................................49
3.2.2.3 Transient transfection of cell lines......................................................49
3.2.2.4 Generation of murine bone marrow derived DCs ..............................50
3.2.3 Virus propagation and titer determination...............50
3.2.3.1 Propagation of VSV strains ................................................................50
3.2.3.2 EMCV stock preparation....51
3.2.3.3 Determination of virus titers by plaque assay....51
3.2.4 Dual Luciferase reporter assay ................................................................52
3.2.5 Stimulation of DCs and determination of cytokine levels by ELISA .....53
3.2.6 Molecular Biology...................................................................................55
3.2.6.1 Isolation and purification of DNA......................55
3.2.6.2 Analysis and cloning of DNA..............................57
3.2.6.3 Site-directed mutagenesis ...................................................................59
3.2.6.4 Synthesis of 5´-triphosphate RNA by in vitro transcription................61
3.2.6.5 Isolation of total RNA from virus-infected cells.63
3.2.6.6 Isolation of total RNA from stimulated DCs and expression analysis of
cytokines and ISGs by quantitative real-time PCR.............................63
II TABLE OF CONTENTS
3.2.6.7 Isolation of RNA from bacteria...........................................................65
3.2.7 Protein biochemistry................................................66
3.2.7.1 Preparation of cell lysates..................................66
3.2.7.2 Determination of protein concentration.............67
3.2.7.3 Immunoprecipitation...........................................67
3.2.7.4 SDS polyacrylamide gel electrophoresis (PAGE) ..............................68
3.2.7.5 Silver staining of SDS-PAGE gels ......................................................69
3.2.7.6 Mass spectrometry analysis................................70
3.2.7.7 Immunoblotting...................................................70
3.2.7.8 RNA binding assay..............................................71
4 Results...................................................73
4.1 Identification of a functional domain at the C-terminus of RIG-I..................73
4.1.1 AA 802 to 925 of RIG-I can be expressed as Flag-tagged protein .........73
4.1.2 Overexpression of RIG-I (AA 802-925) inhibits RIG-I-mediated IFN
induction..................................................................................................74
4.1.3 The C-terminal domain of RIG-I contains conserved cysteine residues
which form a zinc-binding cluster...........................76
4.1.4 The role of C-terminal Cys residues for RIG-I-mediated IFN induction 79
4.1.4.1 IFN-β promoter activation in response to virus infection and
5´-triphosphate RNA...........................................................................79
4.1.4.2 Influence of the integrity of the zinc-binding site in the CTD of RIG-I
on endogenous RIG-I signaling ..........................................................85
4.1.5 Involvement of the zinc-binding site in the CTD of RIG-I in the
interaction with signaling adapter IPS-1 and binding of 5´-triphosphate
RNA.........................................................................................................87
4.1.6 5´-triphosphate RNA interacts with a positively charged conserved
binding groove in the CTD of RIG-I.......................................................89
4.2 The role of the cysteine rich C-terminal domain of MDA5 .............................94