Inhibition of intracellular co-factors for HCMV replication: new strategy for antiviral therapy
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| Format: | Thesis Book |
| Language: | English |
| Published: |
Berlin
2016
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XIV, 147, iv Seiten Illustrationen, Diagramme |
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| 100 | 1 | |a Napierkowski, Ira |d 1986- |e Verfasser |0 (DE-588)1117044564 |4 aut | |
| 245 | 1 | 0 | |a Inhibition of intracellular co-factors for HCMV replication |b new strategy for antiviral therapy |c von Ira Napierkowski |
| 264 | 1 | |a Berlin |c 2016 | |
| 300 | |a XIV, 147, iv Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Humboldt-Universität zu Berlin |d 2016 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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| adam_text | TABLE OF CONTENTS
ZUSAMMENFASSUNG..............................................................................................................................
I
ABSTRACT..................................................................................................................................................ILL
TABLE OF
CONTENTS..............................................................................................................................
IV
LIST OF
FIGURES...................................................................................................................................VIII
LIST OF
TABLES........................................................................................................................................X
LIST OF
ABBREVIATIONS....................................................................................................................
XII
1.
INTRODUCTION...............................................................................................................................1
1.1 THE HUMAN
CYTOMEGALOVIRUS................................................................................................
1
1.1.1 CLASSIFICATION AND EPIDEMIOLOGICAL
ASPECTS..............................................................................1
1.1.2 VIRION AND GENOME
STRUCTURE......................................................................................................3
1.1.3 THE VIRAL LIFE
CYCLE........................................................................................................................
5
1.1.4 MODULATION OF HOST CELL AND IMMUNE RESPONSE BY
HCMV......................................................8
1.1.5 DNA PACKAGING AND
PUL77.........................................................................................................9
1.2 THE YEAST TWO-HYBRID SYSTEM (Y2H SYSTEM)
................................................................
11
1.3 RNA INTERFERENCE
(RNAI).......................................................................................................13
1.4 AIM OF THE
STUDY.....................................................................................................................
15
2. MATERIALS AND
METHODS........................................................................................................16
2.1 M
ATERIALS..................................................................................................................................
16
2.1.1
MACHINES....................................................................................................................................
16
2.1.2 GLASS-, PLASTICWARE AND
MEMBRANES........................................................................................17
2.1.3 CHEMICALS AND
ANTIBIOTICS.........................................................................................................
17
2.1.4
ANTIBODIES...................................................................................................................................
19
2.1.5 COMMERCIAL
KITS.........................................................................................................................
20
2.1.6 ENZYMES AND
LADDERS.................................................................................................................21
2.1.7
OLIGONUCLEOTIDES........................................................................................................................
21
2.1.8
PLASMIDS......................................................................................................................................
23
2.1.9 BUFFERS AND
MEDIA......................................................................................................................
25
2.1.10
ORGANISMS...................................................................................................................................
29
2.2
METHODS...................................................................................................................................
31
2.2.1 MODIFICATION, ANALYSIS AND PURIFICATION OF NUCLEIC
ACIDS.........................................................31
2.2.1.1 GENE AMPLIFICATION BY PCR (POLYMERASE CHAIN REACTION)
....................................................
31
2.2.1.2 MODIFICATION OF DNA WITH RESTRICTION
ENDONUCLEASES........................................................34
2.2.1.3 DEPHOSPHORYLATION OF VECTOR DNA
2.2.1.4 PHOSPHORYLATION OF INSERT DNA
.............................................................................................
35
2.2.1.5
LIGATION......................................................................................................................................36
2.2.1.6 AGAROSE GEL
ELECTROPHORESIS...................................................................................................36
2.2.1.7 PURIFICATION OF
DNA.................................................................................................................
36
2.2.1.8 DETERMINATION OF THE CONCENTRATION OF NUCLEIC
ACIDS.........................................................37
2.2.1.9 CONSTRUCTION OF YEAST BAIT EXPRESSION VECTORS
....................................................................
37
2.2.1.10 CONSTRUCTION OF PREY EXPRESSION VECTORS FOR
CO-IP...........................................................37
2.2.1.11 CONSTRUCTION OF PSIREN-EGFP-RETROQ SHRNA EXPRESSION
VECTORS.................................38
2.2.1.12
SEQUENCING............................................................................................................................
38
2.2.2 ANALYSIS OF PROTEINS AND PROTEIN-PROTEIN
INTERACTIONS.........................................................39
2.2.2.1 DETECTION OF PROTEIN-PROTEIN INTERACTIONS BY
CO-IMMUNOPRECIPITATION (CO-IP)
..............
39
2.22.2 SEPARATION OF PROTEINS BY
SDS-PAGE...................................................................................40
2.2.2.3 DETECTION OF PROTEINS BY
IMMUNOSTAINING...........................................................................40
2.2.2.4 DENSITOMETRIE ANALYSIS OF WESTERN BLOT PICTURES FOR COMPARISON
OF RELATIVE PROTEIN
CONCENTRATIONS........................................................................................................................41
2.2.2.5 DETECTION OF PROTEINS BY COOMASSIE
STAINING.......................................................................42
2.2.3 BACTERIAL CULTURE OF ESCHERICHIA COLI (E.
COLI)..........................................................................
42
2.2.3.1 CULTIVATION OF E.
COLI................................................................................................................42
2.23.2 PRODUCTION OF CACLZ COMPETENT E.
COLI.................................................................................42
2.2.3.3 TRANSFORMATION OF CACH COMPETENT E. COLI BY HEAT
SHOCK..................................................43
2.2.3.4 BACTERIAL COLONY
PCR...............................................................................................................43
2.2.3.5 PLASMID PREPARATION FROM E.
COLI...........................................................................................
43
2.2.3.6 STORAGE OF E.
COLI......................................................................................................................
44
2.2.4 YEAST CULTURE OF SACCHAROMYCES
CEREVISIAE...........................................................................44
2.2.4.1 CULTIVATION OF 5. CEREVISIAE
STRAINS.........................................................................................44
2.2.4.2 LIAC-MEDIATED TRANSFORMATION OF S.
CEREVISIAE...................................................................
44
2.2.4.3 MATING OF 5. CEREVISIAE FOR LIBRARY
SCREENING........................................................................45
2.2.4.4 MEDIUM AND HIGH STRINGENCY SCREENING OF POTENTIAL
INTERACTORS.....................................46
2.2.4.5 YEAST COLONY
PCR.....................................................................................................................
46
2.2.4.6 IDENTIFYING IN-FRAME INTERACTION
PARTNERS............................................................................46
2.2.4.7 PLASMID PREPARATION FROM S.
CEREVISIAE...............................................................................
47
2.2.4.8 PROTEIN EXTRACTION FROM S.
CEREVISIAE....................................................................................47
2.2.4.9 STORAGE OF 5.
CEREVISIAE...........................................................................................................48
2.2.5 CELL
CULTURE..................................................................................................................................48
2.2.5.1 CULTIVATION OF MAMMALIAN
CELLS..............................................................................................48
2.2.5.2 TRANSFECTION OF HEK293T CELLS WITH
TURBOFECT*................................................................48
2.2.5.3 PRODUCTION OF RETROVIRUS FOR DELIVERY OF RNAI CONSTRUCTS
..................................................
48
2.2.5.4 INFECTION OF HELFS FOR RETROVIRAL GENE TRANSFER
...................................................................
49
2.2.5.5 PROTEIN EXTRACTION FROM MAMMALIAN
CELLS...........................................................................
49
2.2.5.6 FIXATION AND STAINING FOR IMMUNOFLUORESCENCE ASSAYS (IFAS)
...........................................
49
2.2.5.7 PHOTOGRAPHIC EVALUATION AND DOCUMENTATION OF
IFAS........................................................50
2.2.5.8 LONG-TERM STORAGE OF MAMMALIAN CELLS
...............................................................................
50
2.2.6 CULTIVATION OF
HCMV..................................................................................................................51
2.2.6.1 PREPARATION OF VIRUS
STOCK....................................................................................................51
2.2.6.2 EXPERIMENTAL INFECTION OF HELF CELLS WITH
HCMV................................................................ 51
2.2.6.3 TITRATION OF HCMV (QUANTIFICATION OF INFECTIOUS HCMV
PARTICLES)....................................52
2.2.7 ELECTRON MICROSCOPY (EM) EMBEDDING AND THIN SECTIONING
.................................................
52
2.2.8 PRODUCTION OF PUL77 SPECIFIC ANTIBODIES IN
CHICKENS........................................................... 53
2.2.8.1 PREPARATION OF DNA-MICROCARRIER COATED CARTRIDGES (GENE GUN
AMMUNITION)
................
54
2.2.8.2 IMMUNISATION OF CHICKENS VIA THE HELIOS GENE G
UN...........................................................55
2.2.8.3 EXTRACTION OF IGY FROM CHICKEN EGG YOLK
...............................................................................
55
2.2.8.4 REMOVAL OF CROSS-REACTING ANTIBODIES FROM IGY
PREPARATION.............................................55
3.
RESULTS...........................................................................................................................................57
3.1 ESTABLISHMENT OF THE YEAST TWO-HYBRID SYSTEM
.............................................................
57
3.1.1 CONSTRUCTION AND VERIFICATION OF YEAST BAIT CLONES
...............................................................
57
3.2 LIBRARY
SCREENING....................................................................................................................59
3.2.1 OVERVIEW OF PERFORMED LIBRARY
SCREENS..................................................................................59
3.2.2 HIGH AND MEDIUM STRINGENCY
SELECTION..................................................................................
60
3.2.3 SEQUENCING OF MEDIUM AND HIGH STRINGENCY LIBRARY CLONES
.................................................
62
3.2.4 RETRANSFORMATION OF LIBRARY CLONES INTERACTING WITH PUL77 INTO
YEAST
...............................
63
3.3 EVALUATION OF IDENTIFIED INTERACTIONS WITH PUL77 IN MAMMALIAN
CELLS....................66
3.3.1 EVALUATION OF INTERACTIONS BY CO-IMMUNOPRECIPITATION IN HEK293T
CELLS
.........................
66
3.3.2 EVALUATION OF THE INTERACTION OF PUL77 AND AKAP9-CT BY I
FA.............................................68
3.3.3 GENERATION OF PUL77 SPECIFIC ANTIBODIES IN
CHICKENS........................................................... 73
3.4 KNOCKDOWN OF PUL77 EXPRESSION BY RNA INTERFERENCE (RNAI)
..................................
78
3.4.1 ESTABLISHMENT OF SHRNA MEDIATED KNOCKDOWN OF PUL77
EXPRESSION.................................78
3.4.2 VIRUS YIELD OF HCMV INFECTED CELLS DURING PUL77
KNOCKDOWN.............................................79
3.4.3 PHENOTYPE OF HCMV INFECTED CELLS DURING PUL77
KNOCKDOWN............................................81
3.5 KNOCKDOWN OF AKAP9 EXPRESSION BY RNA INTERFERENCE (RNAI)
...................................
83
3.5.1 ESTABLISHMENT OF SHRNA MEDIATED KNOCKDOWN OF AKAP9
EXPRESSION...............................83
3.5.2 VIRUS YIELD OF HCMV INFECTED CELLS DURING AKAP9
KNOCKDOWN............................................84
3.5.3 PHENOTYPE OF HCMV INFECTED CELLS DURING AKAP9
KNOCKDOWN...........................................87
4.
DISCUSSION...................................................................................................................................89
4.1 GOING FISHING WITH THE YEAST TWO-HYBRID
SYSTEM......................................................89
4.2 INTERACTION OF PUL77 WITH AKAP9 - WRESTLING WITH PROTEIN DETECTION
.......................
90
4.2.1 DETECTION AND LOCALISATION OF TRANSFECTED AND WT PUL77
...................................................
90
4.2.2 PRODUCTION OF PUL77 SPECIFIC ANTIBODIES IN
CHICKENS...........................................................91
4.2.3 DETECTION OF TRANSFECTED AKAP9-CT AND WT
AKAP9.............................................................92
4.2.4 CO-STAINING OF TRANSFECTED PUL77 AND WT AKAP9
................................................................
93
4.2.5 CO-STAINING OF TRANSFECTED PUL77 AND AKAP9-Q
..................................................................
93
4.3 INTERACTION OF PUL77 WITH AKAP9 - WHAT DOES IT M
EAN?.............................................94
4.3.1 EFFECT OF PUL77 KNOCKDOWN AND ENSUING IMPLICATIONS FOR PUL77
FUNCTION
......................
94
4.3.2 KNOWN FUNCTIONS OF AKAP9
ISOFORMS......................................................................................95
4.3.2.1 FUNCTIONS OF GOLGI-SPECIFIC
AKAP9.......................................................................................
97
4.3.2.2 FUNCTIONS OF CENTROSOMAL
AKAP9.........................................................................................
97
4.3.2.3 SHARED FUNCTIONS OF GOLGI-SPECIFIC AND CENTROSOMAL AKAP9
...........................................
97
4.3.3 EFFECT OF AKAP9 KNOCKDOWN AND IMPLICATIONS OF AKAP9 - PUL77
INTERACTION FOR HCMV
INFECTION......................................................................................................................................99
4.3.3.1 CONTRIBUTION TO AC FORMATION AND
EGRESS...........................................................................
99
4.3.3.2 CONTRIBUTION TO CELL CYCLE SENSING OR
MANIPULATION.........................................................101
4.3.3.3 CONTRIBUTION TO IMMUNE EVASION
...............................................
102
4.4 WHAT S THE (CLINICAL)
USE?.................................................................................................
102
5.
REFERENCES.................................................................................................................................106
APPENDIX....................................................................................................................................................I
CURRICULUM V ITA E
........................................................................................................................................I
ACKNOWLEDGEMENTS...............................................................................................................................
III
DECLARATION OF AUTHORSHIP/ EIGENSTAENDIGKEITSERKLAERUNG
...............................................................
IV
|
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| spelling | Napierkowski, Ira 1986- Verfasser (DE-588)1117044564 aut Inhibition of intracellular co-factors for HCMV replication new strategy for antiviral therapy von Ira Napierkowski Berlin 2016 XIV, 147, iv Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Humboldt-Universität zu Berlin 2016 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029252916&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Napierkowski, Ira 1986- Inhibition of intracellular co-factors for HCMV replication new strategy for antiviral therapy |
| subject_GND | (DE-588)4113937-9 |
| title | Inhibition of intracellular co-factors for HCMV replication new strategy for antiviral therapy |
| title_auth | Inhibition of intracellular co-factors for HCMV replication new strategy for antiviral therapy |
| title_exact_search | Inhibition of intracellular co-factors for HCMV replication new strategy for antiviral therapy |
| title_full | Inhibition of intracellular co-factors for HCMV replication new strategy for antiviral therapy von Ira Napierkowski |
| title_fullStr | Inhibition of intracellular co-factors for HCMV replication new strategy for antiviral therapy von Ira Napierkowski |
| title_full_unstemmed | Inhibition of intracellular co-factors for HCMV replication new strategy for antiviral therapy von Ira Napierkowski |
| title_short | Inhibition of intracellular co-factors for HCMV replication |
| title_sort | inhibition of intracellular co factors for hcmv replication new strategy for antiviral therapy |
| title_sub | new strategy for antiviral therapy |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029252916&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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