Determinants of efficient Hepatitis C Virus RNA replication in cell culture:
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| 003 | DE-604 | ||
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| 008 | 201201s2020 gw a||| m||| 00||| eng d | ||
| 015 | |a 20,H12 |2 dnb | ||
| 016 | 7 | |a 1220364126 |2 DE-101 | |
| 020 | |c Broschur | ||
| 035 | |a (OCoLC)1225085607 | ||
| 035 | |a (DE-599)KXP1735524700 | ||
| 040 | |a DE-604 |b ger |e rda | ||
| 041 | 0 | |a eng | |
| 044 | |a gw |c XA-DE-BW | ||
| 049 | |a DE-355 | ||
| 084 | |a 570 |2 sdnb | ||
| 084 | |a 610 |2 sdnb | ||
| 100 | 1 | |a Schenk, Christian |d 1989- |e Verfasser |0 (DE-588)1161047662 |4 aut | |
| 245 | 1 | 0 | |a Determinants of efficient Hepatitis C Virus RNA replication in cell culture |c presented by M.Sc. Christian H. Schenk |
| 264 | 1 | |a Heidelberg |c [2020] | |
| 300 | |a IV, 140 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruperto Carola University Heidelberg, Germany |d 2020 | ||
| 546 | |a Text Englisch, Zusammenfassung in deutscher und englischer Sprache | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 710 | 2 | |a Universität Heidelberg |0 (DE-588)2024349-2 |4 dgg | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1220364126/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032444569&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-032444569 | |
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| _version_ | 1822127858509676544 |
|---|---|
| adam_text |
TABLE
OF
CONTENTS
ZUSAMMENFASSUNG
.
I
ABSTRACT
.
ILL
1
INTRODUCTION
.
1
1.1
H
EPATITIS
C
V
IRUS
(HCV)
.
1
1.1.1
HISTORICAL
AND
CLINICAL
ASPECTS
OF
HC
V.
1
1.1.2
HC
V
LIFE
CYCLE
.
2
1.1.3
NS5A
.
6
1.2
HCV
REPLICATION
IN
CELL
CULTURE
.
9
1.2.1
CELL
CULTURE
SYSTEMS
.
9
1.2.2
PI4KA
AND
ITS
ROLE
IN
HCVREPLICATION
.
11
1.2.3
ENHANCEDHCVRNA
REPLICATION
IN
CELL
CULTURE
.
13
1.3
A
IM
OF
THIS
STUDY
.
17
2
MATERIAL
&
METHODS
.
18
2.1
M
ATERIAL
.
18
2.1.1
ANTIBODIES
.
18
2.1.2
CHEMICALS,
ENZYMES,
KITS
AND
MISCELLANEOUS
MATERIALS
.
19
2.1.3
CELLS
.
20
2.1.4
OLIGONUCLEOTIDES
.21
2.1.5
PLASMIDS
.
23
2.2
M
ETHODS
.
27
2.2.1
NUCLEIC
ACID
AND
OTHER
STANDARD
METHODS
.
27
2.2.2
CELL
CULTURE
.
29
2.2.3
TRANSFECTION
OF
MAMMALIAN
CELLS
.
30
2.2.4
LUCIFERASE
ASSAY
.31
2.2.5
PROTEIN
ANALYSIS
.
32
2.2.6
IMMUNOPRECIPITATION
AND
MASS
SPECTROMETRY
.
33
2.2.
7
MICROSCOPY
.
34
2.2.8
SUBCELLULAR
FRACTIONATION
AND
LIPIDOMIC
ANALYSIS
.
35
2.2.9
STATISTICAL
ANALYSIS
AND
SOFTWARE
.
36
3
RESULTS
.
37
3.1
P
HOSPHATIDYLINOSITOL
4-K
INASE
ALPHA
INTERACTOME
AND
ITS
ROLE
IN
HCV
REPLICATION
37
3.1.1
IDENTIFICATION
OF
PI4KAS
HOST
NETWORK
.
38
3.1.2
THE
ROLE
OF
PI4KA
INTERACTION
PARTNERS
DURING
HCVREPLICATION
.
41
3.2
C
HARACTERIZATION
OF
A
THREONINE
-
RICH
CLUSTER
IN
NS5A
AND
ITS
CONTRIBUTION
TO
HYPERPHOSPHORYLATION
.
45
3.2.1
MUTATIONAL
ANALYSIS
OF
POTENTIALPHOSPHO-ACCEPTOR
SITES
IN
NS5A
.
47
3.2.2
A
PHOSPHO-SPECIFIC
ANTIBODY
REVEALS
PHOSPHORYLATION
OFT242
EXCLUSIVELY
IN
P58
.
50
3.2.3
SUBCELLULAR
LOCALIZATION
OFNS5A
PHOSPHORYLATED
AT
T242
.
51
3.2.4
IMPACT
OF
KINASE
INHIBITORS
ON
T242
PHOSPHORYLATION
.
53
3.2.5
SEQUENCE
DETERMINANTS
OF
T242
PHOSPHORYLATION
.
54
3.2.6
ASSESSMENT
OFPHOSPHORYLATION
EVENTS
IN
THE
THR-RICH
CLUSTER
FOR
OTHER
HCV
GENOTYPES
.
59
3.3
C
HARACTERIZATION
OF
THE
REPLICATION
COMPETENCE
OF
GLT1
(
GT
I
B
)
.62
3.3.1
RNA
REPLICATION CAPACITY
OF
GLT1
WILDTYPE
IN
CELL
CULTURE
.
62
3.3.2
CELL
CULTURE
ADAPTED
GLT1
.
64
3.3.3
GLT1
WILDTYPE
RNA
REPLICATION
IN
SEC14L2
EXPRESSING
CELLS
.
65
3.3.4
COMBINATION
OFSEC14L2
EXPRESSION
WITH
ADAPTIVE
MUTATIONS
OR
THE
PCI
TREATMENT
.
67
3.3.5
RNA
REPLICATION
CAPACITY
OF
CHIMERIC
GENOMES
BETWEEN
CONI
AND
GLT1
.
69
3.4
M
OLECULAR
INSIGHTS
INTO
THE
MODE
OF
ACTION
OF
SEC14L2
.
73
3.4.1
SEC14L2
AND
LIPID
PEROXIDATION
.
73
3.4.2
IMPACT
OF
SEC14L2
EXPRESSION
ON
OPTIMAL
PCI
COMPOSITION
.
76
3.4.3
SEC14L2
DOES
NOT
SUPPORT
HCVREPLICATION
BY
AFFECTING
THE
PI4PPRODUCTION
.
80
3.4.4
MORPHOLOGY
OF
THE
REPLICATION
ORGANELLES
IN
SEC14L2
EXPRESSING
CELLS
.
81
3.4.5
ANALYSIS
OF
THE
LIPID
COMPOSITION
IN
SEC14L2
EXPRESSING
CELLS
.
84
3.5
F
ULL
-
LENGTH
GLT1
REPLICATION
AND
PARTICLE
PRODUCTION
IN
CELL
CULTURE
.
87
3.5.1
ADAPTATION
OF
FULL-LENGTH
GLT1
WILDTYPE
INFECTIVITY
IN
SEC14L2
EXPRESSING
CELLS
.
87
3.5.2
ADAPTATION
OF
FULL-LENGTH
GLT1
K1846TINFECTIVITY
.
89
4
DISCUSSION
.
93
4.1
T
HE
COMPLEX
INTERPLAY
BETWEEN
PI4KA
AND
NS5A
.
93
4.1.1
PI4KA
RECRUITMENT
TO
THE
SITE
OF
VIRAL
REPLICATION
.
93
4.1.2
THE
IMPACT
OF
PI4KA
ON
NS
5
A
PHOSPHORYLATION
.
95
4.1.3
STIMULATION
OF
PI4KA
ACTIVITY
BY
PHOSPHORYLATED
NS5A
.
97
4.2
C
HARACTERIZATION
OF
A
THREONINE
-
RICH
CLUSTER
IN
NS5A
AND
ITS
CONTRIBUTION
TO
HYPERPHOSPHORYLATION
.
98
4.3
C
ONDITIONS
FOR
EFFICIENT
HCV
RNA
REPLICATION
IN
CELL
CULTURE
.102
4.3.1
REPLICATION
ENHANCING
MUTATIONS
AND
THEIR
LINK
TO
PI4KA
.
102
4.3.2
SEC14L2
CONFERS
ENHANCED
RNA
REPLICATION
OF
HC
V
WILDTYPE
ISOLATES
.
105
4.4
I
NSIGHTS
INTO
THE
MODE
OF
ACTION
OF
SEC14L2
.
106
4.5
C
HARACTERIZATION
OF
A
NOVEL
GENOTYPE
1
B
ISOLATE
.112
4.5.1
GLT1
RNA
REPLICATION
CAPACITY
.
113
4.5.2
DEVELOPMENT
OF
AN
INFECTIOUS
GLT1
CELL
CULTURE
SYSTEM
.
115
4.6
O
UTLOOK
.119
5
BIBLIOGRAPHY.
121
6
SUPPLEMENTARY
DATA
.
133
6.1
A
DAPTIVE
MUTATIONS
IN
NS4B
.
133
6.1.1
IMPACT
OF
DIFFERENT
AMINO
ACID
SUBSTITUTIONS
AT
KI
846
OR
VI
897
ON
CONI
RNA
REPLICATION
.
133
6.1.2
CONI
NS4B-HA
AND
MASS
SPECTROMETRY
ANALYSIS
OF
CONI
WILDTYPE
AND
CONI
K1846T
.
134
62
NS5A
PHOSPHORYLATION
AND
PI4KA
STIMULATION
.
136
6.2.1
REQUIREDPHOSPHO-MIMETIC
ANDPHOSPHO-ABLATANT
AMINO
ACID
SUBSTITUTIONS
IN
NS
5
A
FOR
PI4KA
STIMULATION
.
136
7
APPENDIX
.
137
7.1
A
BBREVIATIONS
.
137
7.2
P
UBLICATIONS
.
138
7.3
P
RESENTATIONS
& P
OSTERS
.
139
7.4
D
ANKSAGUNG
.
140 |
| adam_txt |
TABLE
OF
CONTENTS
ZUSAMMENFASSUNG
.
I
ABSTRACT
.
ILL
1
INTRODUCTION
.
1
1.1
H
EPATITIS
C
V
IRUS
(HCV)
.
1
1.1.1
HISTORICAL
AND
CLINICAL
ASPECTS
OF
HC
V.
1
1.1.2
HC
V
LIFE
CYCLE
.
2
1.1.3
NS5A
.
6
1.2
HCV
REPLICATION
IN
CELL
CULTURE
.
9
1.2.1
CELL
CULTURE
SYSTEMS
.
9
1.2.2
PI4KA
AND
ITS
ROLE
IN
HCVREPLICATION
.
11
1.2.3
ENHANCEDHCVRNA
REPLICATION
IN
CELL
CULTURE
.
13
1.3
A
IM
OF
THIS
STUDY
.
17
2
MATERIAL
&
METHODS
.
18
2.1
M
ATERIAL
.
18
2.1.1
ANTIBODIES
.
18
2.1.2
CHEMICALS,
ENZYMES,
KITS
AND
MISCELLANEOUS
MATERIALS
.
19
2.1.3
CELLS
.
20
2.1.4
OLIGONUCLEOTIDES
.21
2.1.5
PLASMIDS
.
23
2.2
M
ETHODS
.
27
2.2.1
NUCLEIC
ACID
AND
OTHER
STANDARD
METHODS
.
27
2.2.2
CELL
CULTURE
.
29
2.2.3
TRANSFECTION
OF
MAMMALIAN
CELLS
.
30
2.2.4
LUCIFERASE
ASSAY
.31
2.2.5
PROTEIN
ANALYSIS
.
32
2.2.6
IMMUNOPRECIPITATION
AND
MASS
SPECTROMETRY
.
33
2.2.
7
MICROSCOPY
.
34
2.2.8
SUBCELLULAR
FRACTIONATION
AND
LIPIDOMIC
ANALYSIS
.
35
2.2.9
STATISTICAL
ANALYSIS
AND
SOFTWARE
.
36
3
RESULTS
.
37
3.1
P
HOSPHATIDYLINOSITOL
4-K
INASE
ALPHA
INTERACTOME
AND
ITS
ROLE
IN
HCV
REPLICATION
37
3.1.1
IDENTIFICATION
OF
PI4KAS
HOST
NETWORK
.
38
3.1.2
THE
ROLE
OF
PI4KA
INTERACTION
PARTNERS
DURING
HCVREPLICATION
.
41
3.2
C
HARACTERIZATION
OF
A
THREONINE
-
RICH
CLUSTER
IN
NS5A
AND
ITS
CONTRIBUTION
TO
HYPERPHOSPHORYLATION
.
45
3.2.1
MUTATIONAL
ANALYSIS
OF
POTENTIALPHOSPHO-ACCEPTOR
SITES
IN
NS5A
.
47
3.2.2
A
PHOSPHO-SPECIFIC
ANTIBODY
REVEALS
PHOSPHORYLATION
OFT242
EXCLUSIVELY
IN
P58
.
50
3.2.3
SUBCELLULAR
LOCALIZATION
OFNS5A
PHOSPHORYLATED
AT
T242
.
51
3.2.4
IMPACT
OF
KINASE
INHIBITORS
ON
T242
PHOSPHORYLATION
.
53
3.2.5
SEQUENCE
DETERMINANTS
OF
T242
PHOSPHORYLATION
.
54
3.2.6
ASSESSMENT
OFPHOSPHORYLATION
EVENTS
IN
THE
THR-RICH
CLUSTER
FOR
OTHER
HCV
GENOTYPES
.
59
3.3
C
HARACTERIZATION
OF
THE
REPLICATION
COMPETENCE
OF
GLT1
(
GT
I
B
)
.62
3.3.1
RNA
REPLICATION CAPACITY
OF
GLT1
WILDTYPE
IN
CELL
CULTURE
.
62
3.3.2
CELL
CULTURE
ADAPTED
GLT1
.
64
3.3.3
GLT1
WILDTYPE
RNA
REPLICATION
IN
SEC14L2
EXPRESSING
CELLS
.
65
3.3.4
COMBINATION
OFSEC14L2
EXPRESSION
WITH
ADAPTIVE
MUTATIONS
OR
THE
PCI
TREATMENT
.
67
3.3.5
RNA
REPLICATION
CAPACITY
OF
CHIMERIC
GENOMES
BETWEEN
CONI
AND
GLT1
.
69
3.4
M
OLECULAR
INSIGHTS
INTO
THE
MODE
OF
ACTION
OF
SEC14L2
.
73
3.4.1
SEC14L2
AND
LIPID
PEROXIDATION
.
73
3.4.2
IMPACT
OF
SEC14L2
EXPRESSION
ON
OPTIMAL
PCI
COMPOSITION
.
76
3.4.3
SEC14L2
DOES
NOT
SUPPORT
HCVREPLICATION
BY
AFFECTING
THE
PI4PPRODUCTION
.
80
3.4.4
MORPHOLOGY
OF
THE
REPLICATION
ORGANELLES
IN
SEC14L2
EXPRESSING
CELLS
.
81
3.4.5
ANALYSIS
OF
THE
LIPID
COMPOSITION
IN
SEC14L2
EXPRESSING
CELLS
.
84
3.5
F
ULL
-
LENGTH
GLT1
REPLICATION
AND
PARTICLE
PRODUCTION
IN
CELL
CULTURE
.
87
3.5.1
ADAPTATION
OF
FULL-LENGTH
GLT1
WILDTYPE
INFECTIVITY
IN
SEC14L2
EXPRESSING
CELLS
.
87
3.5.2
ADAPTATION
OF
FULL-LENGTH
GLT1
K1846TINFECTIVITY
.
89
4
DISCUSSION
.
93
4.1
T
HE
COMPLEX
INTERPLAY
BETWEEN
PI4KA
AND
NS5A
.
93
4.1.1
PI4KA
RECRUITMENT
TO
THE
SITE
OF
VIRAL
REPLICATION
.
93
4.1.2
THE
IMPACT
OF
PI4KA
ON
NS
5
A
PHOSPHORYLATION
.
95
4.1.3
STIMULATION
OF
PI4KA
ACTIVITY
BY
PHOSPHORYLATED
NS5A
.
97
4.2
C
HARACTERIZATION
OF
A
THREONINE
-
RICH
CLUSTER
IN
NS5A
AND
ITS
CONTRIBUTION
TO
HYPERPHOSPHORYLATION
.
98
4.3
C
ONDITIONS
FOR
EFFICIENT
HCV
RNA
REPLICATION
IN
CELL
CULTURE
.102
4.3.1
REPLICATION
ENHANCING
MUTATIONS
AND
THEIR
LINK
TO
PI4KA
.
102
4.3.2
SEC14L2
CONFERS
ENHANCED
RNA
REPLICATION
OF
HC
V
WILDTYPE
ISOLATES
.
105
4.4
I
NSIGHTS
INTO
THE
MODE
OF
ACTION
OF
SEC14L2
.
106
4.5
C
HARACTERIZATION
OF
A
NOVEL
GENOTYPE
1
B
ISOLATE
.112
4.5.1
GLT1
RNA
REPLICATION
CAPACITY
.
113
4.5.2
DEVELOPMENT
OF
AN
INFECTIOUS
GLT1
CELL
CULTURE
SYSTEM
.
115
4.6
O
UTLOOK
.119
5
BIBLIOGRAPHY.
121
6
SUPPLEMENTARY
DATA
.
133
6.1
A
DAPTIVE
MUTATIONS
IN
NS4B
.
133
6.1.1
IMPACT
OF
DIFFERENT
AMINO
ACID
SUBSTITUTIONS
AT
KI
846
OR
VI
897
ON
CONI
RNA
REPLICATION
.
133
6.1.2
CONI
NS4B-HA
AND
MASS
SPECTROMETRY
ANALYSIS
OF
CONI
WILDTYPE
AND
CONI
K1846T
.
134
62
NS5A
PHOSPHORYLATION
AND
PI4KA
STIMULATION
.
136
6.2.1
REQUIREDPHOSPHO-MIMETIC
ANDPHOSPHO-ABLATANT
AMINO
ACID
SUBSTITUTIONS
IN
NS
5
A
FOR
PI4KA
STIMULATION
.
136
7
APPENDIX
.
137
7.1
A
BBREVIATIONS
.
137
7.2
P
UBLICATIONS
.
138
7.3
P
RESENTATIONS
& P
OSTERS
.
139
7.4
D
ANKSAGUNG
.
140 |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Schenk, Christian 1989- |
| author_GND | (DE-588)1161047662 |
| author_facet | Schenk, Christian 1989- |
| author_role | aut |
| author_sort | Schenk, Christian 1989- |
| author_variant | c s cs |
| building | Verbundindex |
| bvnumber | BV047037433 |
| ctrlnum | (OCoLC)1225085607 (DE-599)KXP1735524700 |
| discipline | Biologie Medizin |
| discipline_str_mv | Biologie Medizin |
| format | Thesis Book |
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| id | DE-604.BV047037433 |
| illustrated | Illustrated |
| index_date | 2024-07-03T16:04:15Z |
| indexdate | 2025-01-24T11:02:19Z |
| institution | BVB |
| institution_GND | (DE-588)2024349-2 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-032444569 |
| oclc_num | 1225085607 |
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| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | IV, 140 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2020 |
| publishDateSearch | 2020 |
| publishDateSort | 2020 |
| record_format | marc |
| spelling | Schenk, Christian 1989- Verfasser (DE-588)1161047662 aut Determinants of efficient Hepatitis C Virus RNA replication in cell culture presented by M.Sc. Christian H. Schenk Heidelberg [2020] IV, 140 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto Carola University Heidelberg, Germany 2020 Text Englisch, Zusammenfassung in deutscher und englischer Sprache (DE-588)4113937-9 Hochschulschrift gnd-content Universität Heidelberg (DE-588)2024349-2 dgg B:DE-101 application/pdf https://d-nb.info/1220364126/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032444569&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schenk, Christian 1989- Determinants of efficient Hepatitis C Virus RNA replication in cell culture |
| subject_GND | (DE-588)4113937-9 |
| title | Determinants of efficient Hepatitis C Virus RNA replication in cell culture |
| title_auth | Determinants of efficient Hepatitis C Virus RNA replication in cell culture |
| title_exact_search | Determinants of efficient Hepatitis C Virus RNA replication in cell culture |
| title_exact_search_txtP | Determinants of efficient Hepatitis C Virus RNA replication in cell culture |
| title_full | Determinants of efficient Hepatitis C Virus RNA replication in cell culture presented by M.Sc. Christian H. Schenk |
| title_fullStr | Determinants of efficient Hepatitis C Virus RNA replication in cell culture presented by M.Sc. Christian H. Schenk |
| title_full_unstemmed | Determinants of efficient Hepatitis C Virus RNA replication in cell culture presented by M.Sc. Christian H. Schenk |
| title_short | Determinants of efficient Hepatitis C Virus RNA replication in cell culture |
| title_sort | determinants of efficient hepatitis c virus rna replication in cell culture |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1220364126/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032444569&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schenkchristian determinantsofefficienthepatitiscvirusrnareplicationincellculture AT universitatheidelberg determinantsofefficienthepatitiscvirusrnareplicationincellculture |
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