Characterisation of covalent and non-covalent interactions of the cellular ubiquitin-homologous protein SUMO with the major immediate-early transactivator IE2p86 of human cytomegalovirus [Elektronische Ressource] / vorgelegt von Anja Monika Berndt

Anja Smith

Sujets

• Biologie

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

Characterisation of covalent and non-covalent interactions
of the cellular ubiquitin-homologous protein SUMO with the
major immediate-early transactivator IE2p86 of human
cytomegalovirus








Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades









vorgelegt von
Anja Monika Berndt
aus Nürnberg





Als Dissertation genehmigt von der Naturwissen-
schaftlichen Fakultät der Universität Erlangen-Nürnberg
















Tag der mündlichen Prüfung: 19. Dezember 2008

Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Eckhart Schweizer

Zweitberichterstatter: Prof. Dr. Michael Mach









I believe in intuition and inspiration.
Imagination is more important than knowledge.
For knowledge is limited, whereas imagination embraces the entire world, stimulating
progress, giving birth to evolution. It is, strictly speaking, a real factor in scientific
research.
Albert Einstein
Table of contents
Table of contents

A. Summary 1

A. Zusammenfassung 2

B. Introduction 3

C. Objectives 11

D. Material and Methods 12
1. Biological materials 12
1.1. Bacterial strains 12
1.2. Yeast strains 12
1.3. Mammalian cell cultures 12
1.4. Virus strains 13
1.5. Antibodies 13
1.5.1. Monoclonal antibodies 13
1.5.2. Polyclonal antibodies 13
1.5.3. Secondary antibodies 14
2. Nucleic acids 14
2.1. Oligonucleotides 14
2.1.1. Primers for the generation of a ds cDNA library 14
2.1.2. Primers for site-directed mutagenesis 14
2.1.3. Primers for sequencing and amplification 14
2.1.4. Primers for quantitative real-time PCR 15
2.2. Provided vectors and plasmids 15
2.2.1. Provided vectors 15
2.2.2. Provided yeast expression plasmids 16
2.2.3. Provided mammalian expression plasmids 17
2.3. Newly constructed plasmids 18
2.4. BACmids 19
2.5. Additional nucleic acids 21
3. Enzymes, chemicals and media 21
3.1. Enzymes 21
3.2. Chemicals 21
3.3. Media 21
3.3.1. Bacterial media 21
3.3.2. Yeast Media 22
3.3.3. Mammalian cell culture media 23
3.4. Standard buffers 23
4. Standard techniques 24
4.1. Site-directed mutagenesis 24
4.2. Generation of a cDNA library for yeast two-hybrid screens 25
5. Cell culture techniques 26
5.1. Yeast cultures 26
5.1.1. Small-scale transformation of yeast 26
5.1.2. Yeast mating 27
5.2. Mammalian cell cultures 28
5.2.1. Maintenance of mammalian cell cultures 28
5.2.2. Transfection of mammalian cells 28
5.2.3. Infection of human foreskin fibroblasts (HFF) 29
6. Indirect immunofluorescence analysis 29
7. Analysis of protein interactions 29
7.1. Luciferase assays 29
Table of contents
7.2. Co-immunoprecipitation 30
7.2.1. Native conditions 30
7.2.2. Denaturing conditions 30
7.3. Protein interactions in yeast 31
7.3.1. Filter-lift assays in yeast 31
7.3.2. ONPG-assays 31
8. Generation and characterisation of recombinant HCMVs 32
8.1. Homologous recombination using linear DNA fragments 32
8.2. Preparation and restriction analysis of BACmid DNA 33
8.3. Reconstitution of infectious viral particles 34
8.4. Quantification of viral genomes by quantitative real-time PCR
(TaqMan-PCR) 34
8.5. Titration by immunofluorescence 35
8.6. Multistep growth curve analysis 35
8.7. Titration by plaque assays 35

E. Results 37
1. Identification of new IE2p86 interaction partners by yeast two-hybrid
assays 37
1.1. Generation of a GAL4 AD fusion cDNA-library for yeast two-hybrid
screening assays using RNA from infected cells 38
1.2. Screening for new IE2p86 interaction partners using the BD SMART ds
cDNA library 39
1.3. Confirmation of identified IE2p86 interaction partners 41

2. Non-covalent and covalent interactions between IE2p86 and SUMO
in yeast and transiently transfected mammalian cells 43
2.1. Identification of a non-covalent SUMO interaction motif within the
exon 5 of IE2p86 43
2.1.1. Mutation of the SUMO interaction motif (SIM) within the exon 5
of IE2p86 43
2.1.2. Analysis of the interaction of SUMO with IE2p86 SIM mutants by
yeast two-hybrid assays 44
2.2. Interaction of IE2p86 with cellular proteins in dependence on the ability
of IE2p86 to interact with SUMO in yeast 46
2.3. Mutation of the SUMO interaction motif within the exon 5 of IE2p86
in mammalian expression vectors 47
2.3.1. Co-immunoprecipitation of Flag-SUMO and IE2p86 from transiently
transfected HEK-293T cells 48
2.3.2. Impaired transactivation capacity of IE2p86 after mutation of the SUMO
interaction motif 50
2.3.3. No effect of the SIM mutation on autorepression of the major immediate
early enhancer/promoter (MIEP) by IE2p86 51
2.4. SUMO modification-dependent interactions of IE2p86 with cellular proteins
in transiently transfected cells 52
2.4.1. Co-immunoprecipitation of Flag-EED with IE2p86 and IE2p86 mutants 52
2.4.2. Co-immunoprecipitation of Flag-hDaxx with IE2p86 and IE2p86 mutants 53
2.4.2.1. Mapping of the IE2p86 interaction domain within hDaxx 54
2.4.2.2. The interaction between IE2p86 and hDaxx was not bridged by DNA binding 56

3. Influence of covalent and non-covalent interactions of SUMO with the viral
regulatory protein IE2p86 during lytic infection 58
3.1. Sequence comparison of the ORF UL122 exon 5 of the HCMV laboratory
strains AD169, Towne and the clinical isolate VR1814 58

Table of contents
3.2. Generation of an IE2p86 SUMOylation-negative recombinant virus based on
the clinical isolate VR1814 59
3.3. Verification and reconstitution of the recombinant FixBac virus containing the
SUMOylation-negative IE2p86 61
3.4. Characterisation of the recombinant viruses AD169ex5-ΔS and FixBacex5-ΔS,
expressing a SUMOylation-negative IE2p86 63
3.4.1. Multistep growth curve analyses revealed an impaired viral replication in
AD169ex5-ΔS compared to wildtype virus 63
3.4.2. Localisation of SUMOylation-negative IE2p86 was not altered during lytic
infection 64
3.4.3. Quantitative real-time PCR revealed an impaired initiation of IE gene expression
in AD169ex5-ΔS infected cells 65
3.4.4. Impaired accumulation of viral DNA in AD169ex5-ΔS and FixBacex5-ΔS
infected cells 67
3.4.5. Reduced plaque formation after infection with recombinant HCMV
FixBacex5-ΔS 68
3.5. Introduction of a mutation of the IE2p86 SUMO interaction motif into the viral
context 69
3.6. Verification and reconstitution of the recombinant viruses containing the
SUMOylation-negative and SIM mutated IE2p86 70
3.7. Characterisation of the recombinant mutant AD169ex5-ΔSM2 virus 72
3.7.1. Multistep growth curve analysis revealed an impaired viral replication of
AD169ex5-ΔSM2 72
3.7.2. Comparison of the replication capacity of AD169ex5-ΔSM2 and AD169ex5-ΔS 73
3.7.3. Localisation of IE2p86 was not altered upon infection with the AD169ex5-ΔSM2
recombinant virus 75
3.7.4. Kinetics of protein expression during viral replication in cells infected
with AD169ex5-ΔSM2 76

4. The formation of IE2-dots and IE2p86 SUMOylation during infection
was independent of ND10 components 78

5. Involvement of IE2p86 SUMOylation in the ubiquitin pathway 80