GB-Virus-C-Proteine interferieren mit der Replikation von HIV-1 [Elektronische Ressource] / vorgelegt von Susan Jung

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Publié le : vendredi 1 janvier 2010
Lecture(s) : 22
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Source : D-NB.INFO/1008557609/34
Nombre de pages : 118
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GB Virus C Proteine
interferieren mit der Replikation von HIV-1



Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.





vorgelegt von

Susan Jung
aus Frankenberg



Als Dissertation genehmigt von der Naturwissenschaftlichen
Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg.















Tag der mündlichen Prüfung: 19.10.2010

Vorsitzender der
Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Bernhard Fleckenstein
Zweitberichterstatter: Prof. Dr. Wolfgang Hillen


GB Virus C Proteins
interfere with the Replication of HIV-1






To my parents,
for their continued support and encouragement.

Contents

1. Abstract ................................................................................................................... 1
2. Zusammenfassung ..................................................................................................2
3. Introduction ............3
3.1. Human Immunodeficiency Virus, the etiologic Agent of AIDS 3
3.1.1. Structure and Genome Organization 4
3.1.2. Replication 5
3.1.3. Transmission, Prevalence, and Etiopathology 6
3.2. GB Virus C, a Pathogen without a Disease 8
3.2.1. Structure and Genome Organization 9
3.2.2. Replication 10
3.2.3. Transmission, Prevalence, and Etiopathology 10
4. Aims ..................................................................................................................... 13
5. Results ................... 15
5.1. A Cell Culture System for GB Virus C 15
5.1.1. A Real-Time RT-PCR to detect GB Virus C RNA 15
5.1.2. Limited Replication of GB Virus C in Human B and T Cell Lines 17
5.1.3. Increased Replication of GB Virus C in Human Lymphocytes 18
5.2. Variability of Clinical GB Virus C Isolates 20
5.2.1. A GB Virus C Sera Bank 20
5.2.2. Differences in the Replication Capacity of Clinical GB Virus C Isolates 20
5.2.3. Sequence Divergence of Clinical GB Virus C Isolates 21
5.3. Interference of GB Virus C with Human Immunodeficiency Virus 22
5.3.1. Classification of Clinical GB Virus C Isolates as HIV-Inhibitory and HIV-Non-Inhibitory 22
5.3.2. HIV Impairment by Expression of GB Virus C Gene Cassettes in a Rhadinovirus Vector System 26
5.3.3 Inhibition of different HIV Replication Steps by GB Virus C Proteins 27
5.4. Identification of the GB Virus C E2 Protein as HIV Entry Inhibitor 29
5.4.1. HIV Suppression by Transfection of RNA encoding the GB Virus C Glycoproteins 29
5.4.2. Expression, Purification, and Characterization of GB Virus C E2(340)-Fc Fusion Proteins 30
5.4.3. Mechanism of GB Virus C E2-mediated HIV Inhibition 35
5.5. Induction of HIV-neutralizing Antibodies by GB Virus C 37
5.5.1. Screening for anti-E2 Antibodies Positive Blood Donors 37
5.5.2. HIV Neutralization by anti-E2 Antibodies 39
5.5.3. Mechanism of HIV Inhibition by anti-E2 Antibodies 42
6. Discussion ............................................................................................................ 47
6.1. Effect of GB Virus C on HIV in Cell Culture 47
6.1.1. Replication Capacity of Clinical of GB Virus C Isolates 47
6.1.2. Inhibition of HIV Replication by Clinical GB Virus C Isolates 48
6.1.3. Induction of beta-Chemokines by Clinical GB Virus C Isolates 49
6.2. Interference of GB Virus C Proteins with HIV Replication 50
6.2.1. Generation of Human T Cell Lines Expressing GB Virus C Proteins 50
6.2.2. Suppression of HIV Replication by Several GB Viru 50
6.2.3. Inhibition of HIV Entry by the GB Virus C E2 Protein 52
6.3. Impairment of HIV Infectivity by anti-E2 Antibodies 53
7. Methods ................................................................................................................ 57
7.1. Escherichia coli 57
7.1.1. Preparation of Chemically Competent E . coli 57
7.1.2. Transformation of Chemically Competent E . coli 57
7.2. Preparation, Enzymatic Manipulation, and Analysis of DNA 58
7.2.1. Purification of DNA 58
7.2.2. Modification of DNA 59
7.2.3. Sequencing of DNA 60
7.3. Preparation, EAnalysis of RNA 60
7.3.1. In V itro Transcription of RNA 60
7.3.2. Isolation of RNA 61
7.3.3. Synthesis of cDNA 61
7.4. Mammalian Cell Culture 61
7.4.1. Isolation of Peripheral and Cord Blood Mononucleated Cells 61
7.4.2. Cultivation of Primary Cells and Cell Lines 62
7.4.3. Cell Proliferation Assay 62
7.4.4. Long-term Storage of Eukaryotic Cells 62
7.5. Transfection of DNA and RNA 62
7.5.1. Calcium Phosphate Transfection 62
7.5.2. Electroporation 64
7.5.3. Selection of Eukaryotic Cells 64
7.6. Expression, Purification, and Analysis of Proteins 64
7.6.1. Protein Expression in Eukaryotic Cells 64
7.6.2. Detection of Proteins 65
7.6.3. Determination of Protein Concentrations 66
7.6.4. Purification of Proteins by Affinity Chromatography 67
7.6.5. Deglycosylation of Proteins 68
7.7. Incubation Assays 68
7.7.1. Incubation of Human T Cells with GBV-C E2-Fc Fusion Proteins 68
7.7.2. Incubation of HIV-1 Pseudoparticles with anti-E2 Antibodies 68
7.7.3. IC 68 50
7.8. Infection Assays 69
7.8.1. GBV-C Infection of Primary Cells and Cell Lines 69
7.8.2. Gene Transfer into Human T Cells by Herpesvirus saimiri 69
7.8.3. HIV-1 Infection Assays 70
7.8.4. Plaque Assays 71
7.9. Immuno Assays 71
7.9.1. Detection of anti-E2 Antibodies 71
7.9.2. Detection of Viral and Recombinant GBV-C E2 Proteins 71
7.9.3. Detection of HIV-1 specific Antibodies 72
7.9.4. Quantification of HIV p24 72
7.9.5. Membrane Lipid Array 72
7.10. Immunohistochemistry 72
7.10.1. Immunofluorescence 72
7.10.2. Flow Cytometry 72
7.10.3. Fluorescence Activated Cell Sorting 72
7.11. Polymerase Chain Reactions 73
7.11.1. Molecular Cloning of Recombinant Proteins 73
7.11.2. Colony PCR 73
7.11.3. Quantification of Viral DNA by Real-Time PCR 73
7.11.4. Viral RNA by Real-Time RT-PCR 74
8. Material ................................................................................................................. 77
8.1. Media, Buffers, and Solutions 77
8.1.1. Media, Buffers, and Solutions for Prokaryotic Cell Culture 77
8.1.2. MedEukaryotic Cell Culture 77
8.1.3. MedProtein Analysis 79
8.2. Cells 81
8.2.1. E scherichia coli 81
8.2.2. Mammalian Cell Lines 82
8.3. Antibodies and Normal Sera 83
8.4. Plasmids 84
8.4.1. GBV-C Expression Plasmids 84
8.4.2. HIV Expression Plasmids 86
8.4.3. Other Expression Constructs 87
8.5. Oligonucleotides 88
8.5.1. GBV-C specific Primers and Probes 88
8.5.2. Other Gene specific Primers and Probes 90
8.6. Kits and Enzymes 90
8.7. Chemicals 91
8.8. Consumables 93
9. Abbreviations ....................................................................................................... 95
10. Bibliography ......... 97
11. Publications ......... 104
13. Acknowledgements ............................................................................................. 105
List of Figures
Figure 3-1: Structure of an HIV particle. ..............................................................................................................................................4
Figure 3-2: HIV replication cycle. ..........................5
Figure 3-3: Unrooted phylogenetic tree of various members of the Flaviviridae. ...........9
Figure 3-4: Seroprevalence of GBV-C. ...............................................................................................................................................11
Figure 5-1: Comparison of different RNA purification methods. .15
®Figure 5-2: Detection of GBV-C RNA using the AbiPrism 7700 and 7000. ............16
Figure 5-3: Replication of GBV-C on human B and T cell lines. ..................................................................................................17
Figure 5-4: GBV-C on PBMC. ...................................18
Figure 5-5: GBV-C on CBL. ......19
Figure 5-6: Correlation of GBV-C virus load and HIV status. .......................................................................................................20
Figure 5-7: Variability of the replication capacity of clinical GBV-C isolates. .............21
Figure 5-8: Mutations in the amino acid sequence of the envelope protein E2 of clinical GBV-C isolates. .........................22
Figure 5-9: Clinical GBV-C isolates differ in their HIV-inhibitory phenotype. ..........................................................................23
Figure 5-10: Inhibition of HIV by purified GBV-C virions. .........................................23 WT
Figure 5-11: by clinical GBV-C isolates. ..........24 PP
Figure 5-12: Down regulation of CCR5 surface expression by HIV-inhibitory GBV-C isolates. .............................................25
Figure 5-13: Chemokine induction by HIV-inhibitory GBV-C isolates. ........................................................25
Figure 5-14: Generation of T cell lines stable expressing different GBV-C genes by infection of HVS . 26 GBV-C
Figure 5-15: Protein expression in HVS transformed T cell lines. ..........................................................27 GBV-C
Figure 5-16: HIV inhibition on HVS transformed T cell lines. ...............................................................27 GBV-C
Figure 5-17: Vectors for eukaryotic expression of GBV-C proteins. ..............................28
Figure 5-18: Expression of GBV-C proteins in CEMx174 Tet-Off cells. ...............................................................................28 CCR5
Figure 5-19: Inhibition of HIV by the GBV-C proteins E2, NS3, and NS5A. ......... 29 PP
Figure 5-20: Inhibition of HIV by transfection of RNA encoding the GBV-C envelope proteins. .................................... 30 WT
Figure 5-21: Model of the GBV-C envelope proteins E1 and E2. ..................................................................31
Figure 5-22: Expression of GBV-C E2(340)-Fc fusion proteins in 293T cells. ............32
Figure 5-23: Characterization of Fc fusion proteins. ..........................................................32
Figure 5-24: Inhibition of HIV and HIV by recombinant GBV-C E2 proteins. ................................................................. 33 WT PP
Figure 5-25: E2(340)-Fc fusion proteins inhibit the gp120/CD4-dependent HIV entry mechanism. .....34
Figure 5-26: Down regulation of CCR5 surface expression by recombinant GBV-C E2 proteins. ..........35
Figure 5-27: Chemokine and cytokine induction by recombinant GBV-C E2 proteins. .............................................................36
Figure 5-28: Recombinant GBV-C E2 proteins bind to human cells. ............................................................37
Figure 5-29: Screening for GBV-C anti-E2 antibodies. .....................................................38
Figure 5-30: Determent of the IgG isotype of human anti-E2 antibodies. ....................................................39
Figure 5-31: Neutralization of HIV by anti-E2 antibodies. ..........................................................................40 WT
Figure 5-32: Neutralization of HIV by anti-E2 antibodies. ...........................................................................41 PP
Figure 5-33: Neutralization ability of anti-E2 antibodies is restricted to HIV. ............................................. 42
Figure 5-34: Neutralization of the anti-E2 antibodies mediated HIV-inhibitory effect. ..............................................................43
Figure 5-35: Immunoprecipitation of HIV by anti-E2 antibodies. ...............................................................43 PP
Figure 5-36: Cell binding by anti-E2 antibodies. .................................................................44
Figure 5-37: Binding of anti-E2 antibodies to phosphatidylinositols. .............................................................45
Figure 7-1: Calculation of the transformation efficiency. ................................................................................................................57
Figure 7-2: Calculation of picomoles of DNA ends. ........................59
Figure 7-3: Calculation of micrograms to picomoles of DNA. ......................................................................................................60
Figure 7-4: pH range of different electrophoresis buffers. ..............66
Figure 7-5: Cytopathic effect of Herpesvirus saimiri on OMK cells. 70
Figure 7-6: Calculation of the TCID . ...............................................................................................................................................71 50

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