Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors
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| Sprache: | English |
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2012
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| 245 | 1 | 0 | |a Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors |c [presented by Dragana Slavkovic Lukic] |
| 264 | 1 | |c 2012 | |
| 300 | |a 119 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
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| 500 | |a Zsfassung in engl. und dt. Sprache | ||
| 502 | |a Heidelberg, Univ., Diss., 2012 | ||
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Datensatz im Suchindex
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|---|---|
| DE-BY-UBR_katkey | 5032003 |
| DE-BY-UBR_location | 00 |
| DE-BY-UBR_media_number | 069037922547 |
| _version_ | 1822675494604111872 |
| adam_text | IMAGE 1
TABLE OF CONTENTS
ZUSAMMENFASSUNG 1
SUMMARY 2
1 INTRODUCTION 3
1.1 RETROVIRUSES 3
1.2 FOAMY VIRUSES 4
1.2.1 GENOME ORGANIZATION AND GENE EXPRESSION 5
1.2.2 THE REPLICATION PATHWAY OF FOAMY VIRUSES 6
1.2.3 TRANSMISSION OF FOAMY VIRUSES 8
1.2.4 FOAMY VIRAL BET PROTEIN 9
1.2.5 FV VECTORS 10
1.3 CELLULAR RESTRICTION FACTORS 11
1.3.1 APOBEC3 12
1.3.2 FELINE APOBEC3 15
1.3.3 RETROVIRAL COUNTER-DEFENSE AGAINST A3-MEDIATED RESTRICTION 16 1.4
AIMS OF THIS STUDY 19
2 MATERIALS AND METHODS 20
2.1 MATERIAL 20
2.1.1 CELLS 20
2.1.1.1 EUKARYOTIC CELLS 20
2.1.1.2 BACTERIAL CELLS 20
2.1.2 PLASMIDS 20
2.1.3 ANTIBODIES/SERA 25
2.1.4 BUFFERS AND SOLUTIONS 26
2.1.5 ENZYMES 27
2.1.6 KITS 27
2.1.7 MEDIA 27
2.1.7.1 CELL CULTURE MEDIUM AND SOLUTIONS 27
2.1.7.2 MEDIA FOR BACTERIAL CULTURE 28
2.1.8 MATERIALS AND DEVICES 28
2.1.9 REAGENTS 29
2.1.10 PRIMERS 31
2.2 METHODS 33
2.2.1 CELL CULTURE AND VIROLOGICAL METHODS 33
2.2.1.1 CELL CULTIVATION AND CRYOPRESERVATION OF CELLS 33
2.2.1.2 INFECTION OF CRFK CELLS 33
2.2.1.3 TRANSFECTION-CAP0 4 33
2.2.1.4 FEFAB TITRATION 34
2.2.2 MICROBIOLOGICAL METHODS 35
2.2.2.1 PREPARATION OF ELECTROCOPMETENT TOPIO CELLS 35
2.2.2.2 ELECTROPORATION 35
2.2.2.3 PRODUCTION OF GST/GST-FEA3 PROTEINS IN E. COLI BI21 35
2.2.3 PROTEIN BIOCHEMICAL ANALYSIS 36
2.2.3.1 PULL-DOWN PROTEIN BINDING ASSAY 36
2.2.3.2 CO-IMMUNOPRECIPITATION 36
2.2.3.3 DETERMINATION OF PROTEIN CONCENTRATION 36
2.2.3.4 SDS POLYACRILAMIDE GEL ELECTROPHORESIS AND IMMUNOBLOTTING 37
2.2.3.5 MEMBRANE STRIPPING 38
2.2.3.6 IMMUNO-FLUORESCENCE AND CONFOCAL MICROSCOPY 38
2.2.4 CLONING AND MUTAGENESIS 39
2.2.4.1 POLYMERASE CHAIN REACTION (PCR) 39
2.2.4.2 FUSION PCR 40
2.2.4.3 PURIFICATION OF PCR PRODUCTS 41
HTTP://D-NB.INFO/102579480X
IMAGE 2
2.2.4.4 RESTRICTION ENZYME DIGESTION OF DNA 41
2.2.4.5 DNA GEL ELECTROPHORESIS 42
2.2.4.6 GEL EXTRACTION 42
2.2.4.7 DNA CONCENTRATION MEASUREMENT 42
2.2.4.8 LIGATION 42
2.2.4.9 DNA SEQUENCING 42
2.2.4.10 COMPUTER ANALYSIS 42
2.2.5 STATISTICAL ANALYSIS 43
2.2.6 DNA PREPARATION 43
2.2.6.1 MINI-PREP 43
2.2.6.2 MAXI-PREP 43
3 RESULTS 44
3.1 FOUR MOTIFS ARE CONSERVED IN BET PROTEINS 44
3.2 BET DELETION MUTANTS 45
3.2.1 N-TERMINAL BET DELETION MUTANTS 45
3.2.1.1 FUNCTIONAL STUDIES 46
3.2.1.2 V5-TAGGED BET N-TERMINAL DELETION MUTANTS 50
3.2.1.3 BINDING STUDIES 52
3.2.2 C-TERMINAL BET DELETION MUTANTS 54
3.2.2.1 FUNCTIONAL STUDIES 54
3.2.2.2 BINDING STUDIES 57
3.2.3 BET-GFP FUSION PROTEINS 58
3.2.3.1 CLONING OF GFP FUSION PROTEINS 58
3.2.3.2 FUNCTIONAL STUDIES 59
3.2.3.3 BINDING STUDIES 61
3.3 CHIMERIC BET PROTEINS 62
3.3.1 CLONING OF CHIMERIC BET PROTEINS 64
3.3.2 FUNCTIONAL STUDIES 65
3.3.3 BINDING STUDIES 68
3.4 SITE DIRECTED MUTAGENESIS OF BET 71
3.4.1 SITE-SPECIFIC ALANINE SCANING MUTAGENESIS OF THE SECOND CONSERVED
MOTIF OF FFV BET 71
3.4.1.1 FUNCTIONAL STUDIES 72
3.4.1.2 BINDING STUDIES 75
3.4.2 SITE-DIRECTED ALANINE SCANNING MUTAGENESIS OF THE FIRST, THIRD AND
THE FOURTH CONSERVED MOTIFS 76
3.4.3 FUNCTIONAL STUDIES 77
3.4.4 BINDING STUDIES 78
3.4.5 THE SECONDARY STRUCTURE OF SUBSTITUTION MUTANTS OF FFV BET 79 3.5
CONCENTRATION-DEPENDENT FEA3Z2B INACTIVATION BY BET 81
3.6 COLOCALIZATION OF WT AND MUTANT BET PROTEINS WITH FEA3S 83
3.7 REPLICATION OF FFV-BBTR IN THE PRESENCE OF NONFUNCTIONAL BET MUTANTS
87 3.8 THERE IS NO TRANS-DOMINANT NEGATIVE EFFECT OF MUTANT BET ON THE
ACTIVITY OF WT BET 88
3.9 DETECTION AND EXPLOITATION OF TRUNCATED FFV BET 90
4 DISCUSSION 96
4.1 THE IMPORTANCE OF BELL AND BEL20RF DOMAINS FOR BET FUNCTION 97 4.1.1
BELL DOMAIN OF BET IS IMPORTANT FOR THE STABILITY OF THE PROTEIN AND NOT
FOR BET FUNCTION 97
4.1.2 THE BEL20RF CONTAINS THE FEA3Z2B BINDING SITE 98
4.1.3 THE C-TERMINUS OF BET PROTEIN IS IMPORTANT FOR THE STABILITY OF
THE PROTEIN AND FOR BINDING TO FEA3Z2B 100
4.2 THE MECHANISM OF FEA3Z2B INACTIVATION BY FFV BET 101
4.3 IMPLICATIONS OF THIS STUDY 103
IMAGE 3
4.3.1 MODE OF BEL20RF EXPRESSION 103
4.3.2 UTILIZATION OF THE N-TERMINAL BELL PROTEIN STABILITY DETERMINANT
FOR VACCINE ANTIGEN EXPRESSION 104
4.3.3 THE IMPORTANCE OF BET FOR FV REPLICATION 104
5 REFERENCES 106
6 ABBREVIATIONS 113
7 TABLE OF FIGURES 116
ACHIEVEMENTS: 119
|
| any_adam_object | 1 |
| author | Slavkovic Lukic, Dragana |
| author_GND | (DE-588)1024916278 |
| author_facet | Slavkovic Lukic, Dragana |
| author_role | aut |
| author_sort | Slavkovic Lukic, Dragana |
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| bvnumber | BV040366357 |
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| dewey-full | 572.8645292569 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.8645292569 |
| dewey-search | 572.8645292569 |
| dewey-sort | 3572.8645292569 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| language | English |
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| physical | 119 Bl. Ill., graph. Darst. |
| publishDate | 2012 |
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| spellingShingle | Slavkovic Lukic, Dragana Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors |
| subject_GND | (DE-588)4113937-9 |
| title | Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors |
| title_auth | Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors |
| title_exact_search | Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors |
| title_full | Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors [presented by Dragana Slavkovic Lukic] |
| title_fullStr | Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors [presented by Dragana Slavkovic Lukic] |
| title_full_unstemmed | Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors [presented by Dragana Slavkovic Lukic] |
| title_short | Characterization of the functional interaction between foamy virus Bet protein and host-encoded APOBEC3 restriction factors |
| title_sort | characterization of the functional interaction between foamy virus bet protein and host encoded apobec3 restriction factors |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025220082&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT slavkoviclukicdragana characterizationofthefunctionalinteractionbetweenfoamyvirusbetproteinandhostencodedapobec3restrictionfactors |