Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus: = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus
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| Format: | Thesis Book |
| Language: | German |
| Published: |
Erlangen ; Nürnberg
2017
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 111 Seiten Illustrationen, Diagramme |
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| 100 | 1 | |a Schilling, Eva-Maria |4 aut | |
| 245 | 1 | 0 | |a Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus |b = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus |c vorgelegt von Eva-Maria Schilling aus Hof (Saale) |
| 246 | 1 | 1 | |a Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus |
| 264 | 1 | |a Erlangen ; Nürnberg |c 2017 | |
| 300 | |a 111 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Friedrich-Alexander-Universität Erlangen-Nürnberg |d 2017 | ||
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| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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| 999 | |a oai:aleph.bib-bvb.de:BVB01-029867338 | ||
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| adam_text | TABLE OF CONTENTS
A. S U M M
ARY...............................................................................................................................
1
A. ZUSAM M
ENFASSUNG.................................................................................................................2
B.
INTRODUCTION.............................................................................................................................
4
1. HUMAN CYTOMEGALOVIRUS
(HCMV)............................................................................................
4
1.1. TRANSMISSION AND
PATHOGENESIS...........................................................................................4
1.2. CLINICAL
MANIFESTATIONS..........................................................................................................
5
1.3. VACCINES AND
THERAPY...........................................................................................................
5
1.4. GENOME STRUCTURE AND VIRION
MORPHOLOGY...........................................................................6
1.5.
REPLICATION.............................................................................................................................
6
2. CELLULAR RESTRICTION
FACTORS........................................................................................................7
2.1.
DEFINITION.................................................................................................................................7
2.2. PML NUCLEAR BODIES
(PML-NBS)...........................................................................................8
2.2.1.
ARCHITECTURE.....................................................................................................................8
2.2.2. ANTIVIRAL
ACTIVITY...............................................................................................................
8
2.2.3. PML AS KEY
COMPONENT.................................................................................................
9
3. ANTAGONIZATION OF PML-NBS BY HCMV EFFECTOR
PROTEINS...................................................11
3.1. MOLECULAR MECHANISM OF IE1-MEDIATED PML ANTAGONIZATION
...........................................
11
C. O
BJECTIVES...............................................................................................................................13
D. MATERIALS AND M ETH OD
S.......................................................................................................
14
1. BIOLOGICAL
MATERIALS..............................................................................................................
14
1.1.
BACTERIA................................................................................................................................14
1.2. YEAST
.......................................
14
1.3. MAMMALIAN CELL
CULTURE.......................................................................................................
14
1.4. VIRUS
STRAIN...........................................................................................................................
14
1.5.
ANTIBODIES............................................................................................................................
15
1.5.1. MONOCLONAL
ANTIBODIES..................................................................................................15
1.5.2. POLYCLONAL
ANTIBODIES....................................................................................................15
1.5.3. SECONDARY ANTIBODIES
16
2. NUCLEIC
ACIDS.............................................................................................................................
16
2.1.
OLIGONUCLEOTIDES..................................................................................................................
16
2.2. VECTORS AND
PLASMIDS...........................................................................................................18
2.2.1. VECTORS AND VECTOR
SYSTEMS..........................................................................................18
2.2.2. PLASMIDS CONTAINING SYNTHETIC GENES
..........................................................................
19
2.2.3. PLASMIDS FISHED IN AN Y2H
SCREEN...............................................................................19
2.2.4. READY-TO-USE
CONSTRUCTS...............................................................................................20
2.2.5. NEWLY GENERATED PLASMIDS
...........................................................................................
22
3. ENZYMES, CHEMICALS, MEDIA, AND BUFFERS
............................................................................
24
3.1.
ENZYMES...............................................................................................................................
24
3.2.
MEDIA....................................................................................................................................
24
3.2.1. MEDIA FOR
YEAST..............................................................................................................24
3.2.2. MEDIATOR
BACTERIA.........................................................................................................
25
3.2.3. MEDIA FOR MAMMALIAN
CELLS..........................................................................................
26
3.3.
CHEMICALS.............................................................................................................................26
3.4. STANDARD BUFFERS AND SOLUTIONS
...........................................................................................
26
4. STANDARD MOLECULAR BIOLOGY
TECHNIQUES...............................................................................28
4.1. SITE-DIRECTED
MUTAGENESIS...................................................................................................
28
4.2. GATEWAY
CLONING..................................................................................................................
28
5. STANDARD CELL CULTURE TECHNIQUES
..........................................................................................
29
5.1. YEAST
CELLS............................................................................................................................29
5.1.1. MAINTENCANCE OF YEAST
CELLS.........................................................................................
29
5.1.2. TRANSFORMATION OF YEAST
CELLS.......................................................................................
29
5.2. MAMMALIAN
CELLS..................................................................................................................
29
5.2.1. MAINTENANCE OF MAMMALIAN
CELLS.................................................................................29
5.2.2. TRANSFECTION OF MAMMALIAN
CELL...................................................................................30
6. INDIRECT IMMUNOFLUORESCENCE ANALYSES
.......................
30
7. ANALYSES OF PROTEIN
INTERACTIONS............................................................................................31
7.1. COIMMUNOPRECIPITATION 31
7.1.1. NATIVE CONDITIONS 31
7.1.2. DENATURING
CONDITIONS...............................................................................................
31
7.2. PROTEIN INTERACTIONS IN
YEAST................................................................................................31
7.2.1. FILTER LIFT
ASSAY..............................................................................................................
31
7.2.2. ONPG
ASSAY.................................................................................................................32
7.3.
GST-PULLDOWN......................................................................................................................
32
8. GENERATION OF STABLY TRANSDUCED CELL
LINES.........................................................................
33
8.1. GENERATION OF LENTIVIRALLY TRANSDUCED CELL
LINES..................................................................33
8.1.1. GENERATION OF INFECTIOUS LENTIVIRAL
PARTICLES.................................................................33
8.1.2. LENTI VIRAL TRANSDUCTION AND SELECTION OF TRANSDUCED
CELLS..........................................34
8.2. GENERATION OF RETROVIRALLY TRANSDUCED CELL LINES
................................................................
34
6.2.1. GENERATION OF INFECTIOUS RETROVIRAL PARTICLES
................................................................
34
6.2.2. RETROVIRAL TRANSDUCTION AND SELECTION OF TRANSDUCED CELLS
.........................................
35
9. INFECTION EXPERIMENTS WITH PRIMARY HUMAN FORESKIN
FIBROBLASTS....................................35
9.1. VIRUS
PREPARATION..................................................................................................................35
9.2. VIRUS
TITRATION........................................................................................................................35
9.3. ANALYSES OF PROTEIN EXPRESSION AND -LOCALIZATION IN HCMV-INFECTED
CELLS......................35
9.4. CHARACTERIZATION OF VIRAL GROWTH BY MULTISTEP GROWTH-CURVE ANALYSIS
..............................
36
10. PROTEIN EXPRESSION AND
PURIFICATION....................................................................................36
10.1. COOMASSIE BLUE
STAINING...................................................................................................36
10.2. LARGE-SCALE INDUCTION OF PROTEIN
EXPRESSION....................................................................36
10.3. PURIFICATION OF PROTEINS BY AFFINITY
CHROMATOGRAPHY........................................................37
10.3.1. GST AFFINITY
CHROMATOGRAPHY.....................................................................................37
10.3.2. NI2+ AFFINITY
CHROMATOGRAPHY......................................................................................37
11. IN VITRO SUMOYLATION
ASSAY.................................................................................................
38
E. R
ESULTS.................................................................................................................................
39
1. DEFINING MINIMAL STRUCTURAL REQUIREMENTS FOR IE1-PML
INTERACTION.............................39
1.1. CHARACTERIZATION OF IE1 DELETION MUTANTS
.......................................................................
39
1.1.1. IE1
CORE
BINDS WITH HIGH AFFINITY TO
PML.......................................................................39
1.1.2. DELETION OF C-TERMINAL A-HELICES LEADS TO A LOSS OF PML BINDING
..............................
40
1.1.3. CHARACTERIZATION OF PRIMARY HUMAN FIBROBLASTS STABLY EXPRESSING
MYC-TAGGED IE1
VERSIONS....................................................................................................................................
43
1.2. CHARACTERIZATION OF PML DELETION AND POINT MUTANTS
...................................................
45
2. THE MECHANISM OF IE1-MEDIATED LOSS OF PML SUMOYLATION
...........................................
48
2.1. IE1 SPECIFICALLY AFFECTS THE SUMOYLATION AT SPECIFIC LYSINE
RESIDUES
...............................
48
2.1.1. IE1 INDUCES THE LOSS OF PML
POLYSUMOYLATION..........................................................48
2.1.2. IE1 SPECIFICALLY AFFECTS THE SUMOYLATION OF PML K160
.............................................
50
2.1.3. IDENTIFICATION OF AT LEAST ONE ADDITIONAL SUMO SITE NOT AFFECTED
BY IE1
....................
52
2.1.4. IE1 PREVENTS ARSENIC TRIOXIDE-MEDIATED PML
DEGRADATION.........................................53
2.2. UNRAVELLING THE MECHANISM OF IE1 -MEDIATED LOSS OF PML
SUMOYLATION........................54
2.2.1. A SUMO MUTANT RESISTANT TO SUMO PROTEASES IS STABLY CONJUGATED TO
PML...........54
2.2.2. GENERATION OF 293 CELLS WITH INDUCIBLE EXPRESSION OF IE 1
..........................................56
2.2.3. IE1 PREVENTS PML DE NOVO SUMOYLATION IN
VIVO........................................................56
2.2.4. PURIFICATION OF THE GST-PML VI/HIS-IE1CORE COMPLEX
................................................
58
2.2.5. IE1 PREVENTS PML DE NOVO SUMOYLATION IN
VITRO.......................................................60
2.2.6. IE1 PREVENTS PML 1-399 DE NOVO SUMOYLATION IN VITRO
............................................
60
2.3. IE1 DOES NOT ACT BY INTERFERING WITH PML
DIMERIZATION......................................................61
3. IDENTIFICATION OF NOVEL IE1 INTERACTION
PARTNERS..................................................................63
3.1. YEAST TWO-HYBRID
SCREEN......................................................................................................
63
3.2. FURTHER EXAMINATION OF SELECTED
INTERACTORS.......................................................................64
4. THE ROLE OF THE NOVEL IE1 INTERACTOR FEN1 FOR HCMV
REPLICATION.....................................65
4.1. FEN1 176-380 INTERACTS WITH
IE 1 C
ORE
AS WELL AS IE1 IN MAMMALIAN CELLS......................65
4.2. FEN1 INTERACTS WITH
IE 1C
ORE
IN MAMMALIAN CELLS
.............................................................
67
4.3. FEN1 IS UPREGULATED DURING HCMV
INFECTION....................................................................66
4.4. IE1 IS NOT SUFFICIENT FOR THE UPREGULATION OF FEN1
EXPRESSION.........................................69
4.5. GENERATION OF PRIMARY HUMAN FORESKIN FIBROBLASTS STABLY EXPRESSING
MCHERRYFENI ....69
4.6. FEN1 LOCALIZES AT DIFFERENT SITES DURING THE INFECTION CYCLE
.............................................
70
4.7. OVEREXPRESSION OF FEN1 LEADS TO A SLIGHTLY ENHANCED EARLY AND LATE
GENE EXPRESSION 72
4.8. DECREASED VIRAL REPLICATION ON FEN1 KNOCKDOWN
HFFS...................................................74
4.9. IE1 BINDING DOES NOT DISRUPT THE INTERACTION BETWEEN FEN1 AND PCNA
.........................
75
F. DISCUSSION 77
1. DEFINING MINIMAL STRUCTURAL REQUIREMENTS FOR IE1-PML INTERACTION
...............................
77
2. THE MECHANISM OF IE1-MEDIATED LOSS OF PML SUMOYLATION
...........................................
81
3. THE ROLE OF THE NOVEL IE1 INTERACTOR FEN1 FOR HCMV
REPLICATION......................................84
G. A
BBREVIATIONS........................................................................................................................
89
H. R
EFERENCES..............................................................................................................................90
I. A
PPENDIX................................................................................................................................
108
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| language | German |
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| spelling | Schilling, Eva-Maria aut Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus vorgelegt von Eva-Maria Schilling aus Hof (Saale) Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus Erlangen ; Nürnberg 2017 111 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Friedrich-Alexander-Universität Erlangen-Nürnberg 2017 Zelluläre Immunität (DE-588)4197656-3 gnd rswk-swf Cytomegalie-Virus (DE-588)4238363-8 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Cytomegalie-Virus (DE-588)4238363-8 s Zelluläre Immunität (DE-588)4197656-3 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029867338&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schilling, Eva-Maria Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus Zelluläre Immunität (DE-588)4197656-3 gnd Cytomegalie-Virus (DE-588)4238363-8 gnd |
| subject_GND | (DE-588)4197656-3 (DE-588)4238363-8 (DE-588)4113937-9 |
| title | Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus |
| title_alt | Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus |
| title_auth | Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus |
| title_exact_search | Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus |
| title_full | Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus vorgelegt von Eva-Maria Schilling aus Hof (Saale) |
| title_fullStr | Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus vorgelegt von Eva-Maria Schilling aus Hof (Saale) |
| title_full_unstemmed | Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus vorgelegt von Eva-Maria Schilling aus Hof (Saale) |
| title_short | Mechanism of deSUMOylation by the regulatory protein IE1 of human cytomegalovirus |
| title_sort | mechanism of desumoylation by the regulatory protein ie1 of human cytomegalovirus funktionsmechanismus der de sumoylierung durch das regulatorprotein ie1 des humanen cytomegalovirus |
| title_sub | = Funktionsmechanismus der de-SUMOylierung durch das Regulatorprotein IE1 des humanen Cytomegalovirus |
| topic | Zelluläre Immunität (DE-588)4197656-3 gnd Cytomegalie-Virus (DE-588)4238363-8 gnd |
| topic_facet | Zelluläre Immunität Cytomegalie-Virus Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029867338&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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