Structural and functional analysis of the hepatitis C virus non-structural protein 5A [Elektronische Ressource] / vorgelegt von Nicole Appel

Nicole Topin

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• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2005
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Structural and Functional Analysis
of the Hepatitis C Virus
Non-Structural Protein 5A


Dissertation zur Erlangung des Grades
Doktor der Naturwissenschaften

Am Fachbereich Biologie
der Johannes Gutenberg-Universität Mainz

vorgelegt von
Nicole Appel
geb. 21.05.1974 Bad Schwalbach























Dekan: Prof. Dr. H. Paulsen
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 16.12.2004

Summary....................................................................................................................................... .7

1. Introduction................................................................................................................................9
1.1 History of Hepatitis Viruses..9
1.2 Genomic organization of HCV............................12
1.3 The HCV non-strucutral protein 5A ....................................................................................15
1.4 HCV Replication cycle: a hypothetical model....................................................................17
1.5 Experimental systems ..........................................18
1.6 Subject of the Thesis............22

2. Materials and Methods............................................................................................................23
2.1 Materials ..............................................................23
2.1.1 Viruses..........................................................................................23
2.1.2 Cells23
2.1.3 Media ............................................................23
2.1.4 Antisera.........................................................................................24
2.1.5 Vectors..........................24
2.1.6 Primer sequences ..........................................................................................................25
2.1.7 Buffers and solutions....26
2.2 Nucleic acid standard methods ............................................................................................27
2.2.1 Isolation of DNA..........................................27
2.2.2 Agarose gel electrophoresis..........................28
2.2.3 DNA extraction from agarose gels ...............................................28
2.2.4 Ligation of DNA-fragments.........................................................28
2.2.5 Transformation of E. coli..............................................................29
2.2.6 Restriction analysis of DNA.........................29
2.2.7 Purification and precipitation of DNA .........................................................................29
2.2.8 Polymerase Chain Reaction (PCR)...............30
2.2.9 PCR-based mutagenesis ................................................................................................31
2.2.10 DNA sequencing analysis...........................31
2.2.11 Preparation of total RNA from cells ...........................................................................32
2.2.12 Quantification of DNA and RNA with absorption spectroscopy...............................32
2.2.13 Reverse transcription of RNA and amplification of c-DNA (RT-PCR).....................33 2.2.14 In vitro transcription ...................................................................................................33
2.2.15 RNA-formaldehyde-gel electrophoresis.....34
2.2.16 RNA-transfection and selection of G418-resistant cell lines.....34
2.2.17 Transient replication assay .........................................................................................35
2.2.18 Glyoxal agarose gel electrophoresis and Northern-hybridization..............................35
2.2.19 Transfection of Huh-7/T7 cells with Lipofectamine 2000 .........................................36
2.3 Expression and Analysis of Proteins ...................................................37
2.3.1 SDS-polyacrylamide-gel electrophoresis (SDS-Page).................37
2.3.2 Western blot..................................................................................37
2.3.3 Vaccinia virus (VV) T7-expression system..................................................................38
2.3.4 Immunoprecipitation.....................................38
2.3.5 Immunofluorescence analysis (IF) ...............................................................................39

3. Results .......................................................................40

3.1 Identification of highly adaptive mutations in NS5A and improvement of the HCV
replicon system.............................................................................................................................40
3.1.1 Identification of cell culture-adapted HCV replicon RNAs.............................................40
3.1.2 Determination of adaptive mutations in the HCV coding sequence.42
3.1.3 Lack of correlation between the ECF and replicon RNA copy number in G418-selected
cell lines .....................................................................................................................................44
3.1.4 Transient replication of HCV replicons carrying a luciferase reporter gene ....................45
3.1.5 Construction of subgenomic replicons with alternative selection markers......................49
FIG. 11:......................................................................................................................................50
3.1.6 Construction and characterization of genomic replicons..................50

3.2 Role of NS5A phosphorylation for RNA replication..........................................................52
3.2.1 Alanine substitutions for highly conserved serine residues in cluster 1 of NS5A and their
effects on hyperphosphorylation and RNA replication.............................52
3.2.2 Combination and deletion of alanine substitutions for highly conserved serine residues of
cluster 1......................................................................................................................................55
3.2.3 Glutamic acid substitutions for highly conserved serine residues in cluster 1 of NS5A
and their effects on hyperphosphorylation and RNA replication..............................................57

3.2.4 Influence of adaptive mutations in NS3, NS4B and NS5B on NS5A
hyperphosphorylation................................................................................................................59
3.2.5 Replication and phosphorylation analysis of cluster 3 located at the C-terminus of NS5A
and involved in basal phosphorylation......................61
3.2.6 Analysis of the genetic flexibility of the C -terminus of NS5A by introduction of
heterologous sequences .............................................................................................................65

3.3 Structural and functional analysis of the NS5A membrane anchor domain...................69
3.3.1 Subcellular localization of NS5A membrane anchor mutants..........................................70
3.3.2 RNA replication of NS5A membrane anchor mutants.....................72
3.3.3 Phosphorylation analysis of NS5A helix mutants............................................................73
3.3.4 Influence of N -terminal NS5A peptides on replication efficiency of subgenomic
replicons.....................................................................................................................................74

3.4 Rescue of HCV RNA replication by trans-complementation of NS5A.............................78
3.4.1 Establishment of a trans-complementation assay using selectable replicons ...................78
3.4.2 Trans-complementation analysis of mutations in NS3, NS4B, NS5A and NS5B............80
3.4.3 Establishment of a transient trans-complementation assay..............................................84
3.4.4 Trans-complementation analysis of mutations in the N-terminal amphipathic a-helix of
NS5A.........................................................................................................89
3.4.5 Trans-complementation analysis of NS5A mutants by non-replicating helper RNA ......90
3.4.6 Mapping of the minimal sequence necessary and sufficient for trans-complementation.92
3.4.7 Trans-complementation analysis of an NS5A mutant by helper RNAs carrying mutations
in NS3, NS4A and NS4B...........................................................................................................93

3.5 Identification and characterization of a novel cellular interaction partner of the
hepatitis C virus NS5A protein...................................................................................................98
3.5.1 Identification of NS5A-interacting phosphoproteins........................98
3.5.2 Identification of the amphiphysin II binding site in NS5A ............................................100
3.5.3 Interaction of NS5A and amphiphysin II in Huh-7 replicon cells..101
3.5.4 Influence of NS5A-amphiphysin II interaction on RNA replication..............................103


4. Discussion...................................