Quantitative analysis of the hepatitis C-virus replication complex and identification of associated cellular factors [Elektronische Ressource] / presented by Doris Quinkert

Doris Hayman

Sujets

• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences








presented by
Diplom-Biologin Doris Quinkert
born in Heidelberg, Germany

Oral-examination:







Quantitative analysis of the Hepatitis C Virus
replication complex and identification of associated
cellular factors











Referees: Prof. Dr. Ralf Bartenschlager
Prof. Dr. Irmgard Sinning
TABLE OF CONTENTS I

Table of contents
Table of contents......................................................................................................... I
Acknowledgements .................................................................................................... V
Summary ................................................................................................................... VI
Zusammenfassung ................................................................................................... VII
1. Introduction.......................................................................................................... 1
1.1 Hepatitis C virus overview ............................................................................ 1
1.2 Course of disease and therapy .................................................................... 2
1.3 Molecular organization of HCV..................................................................... 3
1.3.1 Genetic diversity.................................................................................... 3
1.3.2 Genome organization and function of viral proteins .............................. 4
1.4 Model systems to study HCV replication in cell culture ................................ 7
1.4.1 Tissue culture models ........................................................................... 7
1.5 HCV life cycle............................................................................................. 10
1.6 The HCV RNA Replication Complex .......................................................... 12
1.7 Known cellular cofactors of HCV replication............................................... 15
1.8 Aim of thesis............................................................................................... 17
2. Materials and Methods ...................................................................................... 18
2.1 Materials..................................................................................................... 18
2.1.1 Cells .................................................................................................... 18
2.1.2 Media .................................................................................................. 19
2.1.3 Antibodies and antisera....................................................................... 19
2.1.4 Vectors................................................................................................ 21
2.1.5 Oligonucleotides.................................................................................. 22
2.1.6 siRNAs 23
2.1.7 Buffers and Solutions .......................................................................... 23
2.2 Preparation, analysis, and manipulation of nucleic acids ........................... 25
2.2.1 Plasmid DNA isolation......................................................................... 25
2.2.2 Agarose gel electrophoresis................................................................ 25
2.2.3 DNA extraction from agarose gels ...................................................... 26
2.2.4 Phosphorylation and dephosphorylation of DNA................................. 26
2.2.5 Ligation of DNA-fragments.................................................................. 26
2.2.6 Transformation of E. coli ..................................................................... 26 TABLE OF CONTENTS II

2.2.7 Analysis of DNA with restriction enzymes ........................................... 27
2.2.8 Purification and precipitation of DNA................................................... 27
2.2.9 Polymerase Chain Reaction (PCR)..................................................... 27
2.2.10 Site directed mutagenesis................................................................... 28
2.2.11 DNA sequencing analysis 28
2.2.12 Transfection of cells with plasmid DNA ............................................... 29
2.2.13 Quantification of DNA and RNA with absorption spectroscopy ........... 29
2.2.14 In vitro transcription............................................................................. 29
2.2.15 RNA transfection by electroporation.................................................... 30
2.2.16 RNA formaldehyde gel electrophoresis............................................... 30
2.2.17 Total RNA isolation from eukaryotic cells............................................ 31
2.2.18 RNA glyoxal gel electrophoresis ......................................................... 31
2.2.19 Northern blot analysis ......................................................................... 31
2.2.20 HCV RNA quantification by RT-PCR................................................... 32
2.3 Expression, purification, and analysis of proteins....................................... 33
2.3.1 Isolation of HCV replication complexes............................................... 33
2.3.2 Sucrose density gradient centrifugation .............................................. 33
2.3.3 Bradford assay for protein quantification............................................. 33
2.3.4 SDS polyacrylamide gel electrophoresis (SDS-PAGE) ....................... 34
2.3.5 Western blot........................................................................................ 34
2.3.6 Proteinase K, S7 nuclease and Triton X-100 treatment of CRCs........ 34
2.3.7 TCA precipitation................................................................................. 35
2.3.8 2-dimensional gel electrophoresis....................................................... 35
2.3.9 Silver staining of proteins after SDS-PAGE ........................................ 36
2.3.10 HCV replicase activity assay............................................................... 36
352.3.11 Metabolic S-labeling of proteins 37
2.3.12 Immunoprecipitation............................................................................ 37
2.3.13 Transient silencing by siRNA .............................................................. 38
2.3.14 Immunofluorescence analysis (IF) ...................................................... 38
2.3.15 Flow cytometry analysis ...................................................................... 38
2.3.16 Luciferase assay ................................................................................. 39
2.3.17 Competence induction and transformation of yeast cells .................... 39
2.3.18 Y2H β-galactosidase filter assay......................................................... 39
2.3.19 Quantitative detection of HCV core protein ......................................... 40 TABLE OF CONTENTS III

2.4 Working with viruses .................................................................................. 40
2.4.1 Lentiviral transduction of cells ............................................................. 40
2.4.2 Preparation of Hepatitis C virus stocks ............................................... 41
2.4.3 HCV infection of cells .......................................................................... 41
3. Results .............................................................................................................. 42
3.1 Development of purification strategies for active viral replication complexes
from HCV subgenomic replicon cells .................................................................... 42
3.1.1 Establishment of a replicase activity assay for purified HCV replication
complexes ......................................................................................................... 42
3.1.2 CRC activity is different from polymerase activity ............................... 44
3.1.3 Purification of HCV replication complexes by sucrose floatation gradient
centrifugation..................................................................................................... 46
3.1.4 Purification of HCV replication complexes by detergent treatment...... 48
3.2 Identification of Cellular Factors Associated with the Hepatitis C Virus
Replication Complex ............................................................................................. 52
3.2.1 Comparative 2-dimensional gel analysis of CRCs from replicon cells
and naïve cells .................................................................................................. 55
3.2.2 Identification of cellular proteins associated with HCV replication
complexes by mass spectrometry ..................................................................... 56
3.3 Analysis of the role of Annexin II in HCV replication................................... 61
3.3.1 Colocalization studies of Annexin II with viral pro