The species specificity of CD81 receptor usage by hepatitis C virus [Elektronische Ressource] / Julia Bitzegeio. TWINCORE, Abteilung für Experimentelle Virologie der Medizinische Hochschule Hannover. Betreuer: Thomas Pietschmann

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• Publié par: medizinische_hochschule_hannover_-mhh
• Publié le: 01 janvier 2010
• Nombre de lectures: 32
• Langue: English
• Poids de l'ouvrage: 3 Mo

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Medizinische Hochschule Hannover
TWINCORE, Abteilung für Experimentelle Virologie




The Species Specificity of CD81 Receptor Usage
by Hepatitis C Virus



INAUGURAL - DISSERTATION
zur Erlangung des Grades einer Doktorin
der Naturwissenschaften
- Doctor rerum naturalium -
( Dr. rer.nat. )






vorgelegt von

Julia Bitzegeio

Bad Neuenahr – Ahrweiler


Hannover 2010 Wissenschaftliche Betreuung: Prof. Dr. Thomas Pietschmann
TWINCORE, Hannover
Abteilung für Experimentelle Virologie

Wissenschaftliche Betreuung: Prof. Dr. Stefan Pöhlmann
Medizinische Hochschule Hannover
Instiu fürVirolgie





1. Erst-Gutachterin / Gutachter: Prof. Dr. Thomas Pietschmann
TWINCORE, Hannover
Abteilung für Experimentelle Virologie

2. Gutachterliche Stellungnahme durch: Prof. Dr. Stefan Pöhlmann
Medizinische Hochschule Hannover
Instiu fürVirolgie

3. Gutachterin / Gutachter: Prof. Dr. Beate Sodeik Hochschule Hannover nstiu fürVirolgie





Tag der mündlichen Prüfung: 2. Juli 2010 Acknowledgement I

Acknowledgement

My special thanks go to Prof. Dr. Thomas Pietschmann for giving me the chance to work on
this very interesting project within his department. I want to thank him for his excellent
supervision, the great support and continuous motivation over the years of my thesis.

I also would like to thank Prof. Dr. Stefan Pöhlmann and Prof. Dr. Beate Sodeik for agreeing
to evaluate my thesis as a co-referee.

Special thanks go to Dorothea Bankwitz, who became a great friend and a good colleague. It
was a big pleasure to work with her in a team. I want to thank her for the many discussions
and motivation within and outside of the lab. Furthermore, I like to thank Kathrin Hüging. It
was a pleasure to work and share a bench with her. I am grateful to all members of the
department of experimental virology for the fruitful discussions, the great working
atmosphere and the everlasting supply with sweets and cake.

I want to thank Prof. Dr. Ralf Bartenschlager and all the members of the department of
Molecular Virology in Heidelberg for giving me the opportunity to work in his department
throughout the first year of my thesis.

Finally, my greatest thanks go to my family my parents, my sister and my brother as well as
my friends for their continuous support, motivation and encouragement during my whole
studies.





Table of contents II

Acknowledgement ....................................................................................................................... I
Table of contents ........................................................................................................................II
Summary ................................................................................................................................... V
Zusammenfassung ................................................................................................................... VII
1 Introduction ......................................................................................................................... 1
1.1 Hepatitis C Virus ......................................................................................................... 1
1.2 Organization of the viral genome and the viral proteins ............................................. 2
1.3 Tools to study the HCV life cycle in cell culture ........................................................ 4
1.4 HCV life cycle ............................................................................................................. 5
1.5 HCV cell entry7
1.5.1 The virus particle and the glycoproteins .............................................................. 7
1.5.2 Receptors involved in HCV entry ........................................................................ 9
1.5.3 HCV entry pathway ............................................................................................ 12
1.6 Species specificity of HCV infection13
1.7 Animal models to study HCV infection in vivo ........................................................ 14
1.8 Aim of the thesis ........................................................................................................ 16
2 Materials and Methods ...................................................................................................... 17
2.1 Materials .................................................................................................................... 17
2.1.1 Cells17
2.1.2 Media .................................................................................................................. 19
2.1.3 Antibodies and antisera ...................................................................................... 20
2.1.4 Oligonucleotides ................................................................................................. 21
2.1.5 Vectors ............................................................................................................... 23
2.1.6 siRNAs28
2.1.7 Buffers and Solutions ......................................................................................... 29
2.2 Preperation, analysis and manipulation of DNA and RNA ....................................... 31
2.2.1 Transformation of E.coli .................................................................................... 31 Table of contents III

2.2.2 Plasmid DNA isolation ....................................................................................... 31
2.2.3 Quantification of DNA and RNA with absorption spectroscopy ....................... 32
2.2.4 Total RNA isolation from eukaryotic cells or tissue .......................................... 32
2.2.5 Generation of cDNA .......................................................................................... 32
2.2.6 Polymerase Chain reaction (PCR) ...................................................................... 32
2.2.7 Site directed mutagenesis ................................................................................... 33
2.2.1 Analysis of DNA with restriction enzymes ........................................................ 34
2.2.2 Dephosphorylation of DNA ............................................................................... 34
2.2.3 Agarose gel electrophoresis34
2.2.4 Ligation of DNA-fragments34
2.2.5 DNA sequencing analysis .................................................................................. 35
2.2.6 RNA in vitro transcription35
2.2.7 RNA quantification by RT-PCR ........................................................................ 35
2.2.8 Cloning of adaptive mutations ........................................................................... 36
2.3 Expression, analysis and purification of proteins ...................................................... 37
2.3.1 Cell culture ......................................................................................................... 37
2.3.2 Transfection of cells with Plasmid DNA ........................................................... 37
2.3.3 RNA transfection by electroporation ................................................................. 38
2.3.4 Protein quantification by Bradford assay39
2.3.5 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .................................... 39
2.3.6 Western blot ....................................................................................................... 39
2.3.7 Gene silencing by siRNAs ................................................................................. 40
2.3.8 Immunofluorescence analysis (IF) ..................................................................... 40
2.3.9 Determination of cell surface expression by Flow cytometry analysis .............. 41
2.3.10 Cell sorting by Flow cytometry (FACS) ............................................................ 41
2.3.11 Luciferase assay ................................................................................................. 42
2.3.12 Quantitative detection of core protein ................................................................ 42 Table of contents IV

2.3.13 Purification of GST-tagged proteins from bacteria ............................................ 42
352.3.14 Metabolic S-labelling of HCV glycoproteins .................................................. 43
2.3.15 Immunoprecipitation (IP) of HCV glycoproteins or HCV particles .................. 43
2.4 Working with viruses ................................................................................................. 45
2.4.1 Generation of stable cell lines by lentiviral gene transfer .................................. 45
2.4.2 Preparation of HCV stocks ................................................................................. 45
2.4.3 HCV infection of cells ........................................................................................ 46
2.4.4 HCV histochemistry and determination of virus titers ................................