Analysis of the role of the cellular subnuclear structure ND10 for human cytomegalovirus replication [Elektronische Ressource] / vorgelegt von Nina Tavalai

Natavala

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• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Analysis of the role of the cellular subnuclear structure ND10
for human cytomegalovirus replication











Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades












vorgelegt von
Nina Tavalai
aus Braunschweig



Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg




















Tag der mündlichen Prüfung: 20.03.2008

Vorsitzender der Promotionskommission: Prof. Dr. E. Bänsch

Erstberichterstatter: Prof. Dr. B. Fleckenstein

Zweitberichterstatter: PD Dr. R. Slany

Drittberichterstatter: Prof. Dr. M. Reddehase, Mainz



By three methods we may learn wisdom:
First, by reflection, which is noblest;
Second, by imitation, which is easiest;
and third by experience, which is the bitterest.

Confucius

Table of contents
Table of contents

A. Sumary 1
A. Zusammenfassung 2
B. Introduction 3
C. Objectives 11
D. Materials and Methods 12
1. Biological Materials
1.1. Bacteria 12
1.2. Eukaryotic cell cultures
1.3. Virus strains 12
1.4. Antibodies 12
1.4.1. Monoclonal antibodies
1.4.2. Polyclonal 13
1.4.3. Secondary

2. Nucleic acids 14
2.1. Oligonucleotides
2.2. Vectors and plasmids 15
2.2.1. Vectors and vector systems 15
2.2.2. Ready-to-use DNA constructs 16
2.2.3. Newly generated plasmids and BACmids 17

3. Enzymes, chemicals, and media 18
3.1 Enzymes 18
3.2. Media 19
3.2.1. Bacterial media
3.2.2. Cell culture 19
3.3. Chemicals
3.4. Standard buffers and solutions 19

4. Standard molecular biology techniques 20
4.1. In vitro mutagenesis 21

5. Cell culture techniques
5.1. Maintenance of cell cultures 21
5.2. Transfection and infection

6. Immunofluorescence analysis 22
6.1. Indirect immunofluorescence analysis 22
6.2. Fluorescence in situ hybridization (FISH) 22

7. Time-lapse microscopy of live cells 23

8. Generation of retrovirally transduced cell lines 24
8.1. Generation of infectious retroviral particles 24
8.2. Retroviral transduction and selection of stably transduced cells 25

9. Generation and characterization of recombinant viruses 25
9.1. Generation of recombinant viruses using the BACmid technology 25
9.1.1. Homologous recombination using linear DNA fragments 26
9.2. Isolation and restriction enzyme digestion of BACmid DNA 26
9.3. Southern blot analysis with biotinylated DNA probes 27
9.4. Reconstitution of recombinant viruses 27
9.5. Virus titration 28
9.6. Characterization of recombinant viruses 28
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9.6.1. Multistep growth curve analysis 28
9.6.2. Quantitative real-time PCR (TaqMan-PCR) 29
9.7. Purification of viral particles 29

E. Results 30
1. Examination of the effect of ND10 domains and its components PML and
hDaxx on human cytomegalovirus infection 30
1.1. Generation of primary human fibroblasts with a stable knockdown
of PML or hDaxx 30
1.1.1. Analysis of the subnuclear localization of hDaxx and Sp100 after
depletion of the ND10 scaffold protein PML 31
1.2. Investigation of human cytomegalovirus replication in PML- and
hDaxx-kd cells 33
1.2.1. Evidence for an enhanced replication of HCMV in the absence of PML
or hDaxx 33
1.2.2. Loss of PML or hDaxx results in augmented HCMV IE gene expression 33
1.3. Exclusion of off-target RNAi effects of the selected shRNAs siPML2 and
siDaxx1 35
1.3.1. Generation of a silencing-resistant variant of PML isoform VI 35
1.3.1.1. Reintroduction of PML isoform VI into PML-depleted cells 36
1.3.2. Reconstitution of hDaxx expression in siDaxx1 cells via overexpression
of the ND10 factor 37
1.4. Countermeasures of HCMV against the antiviral effect of ND10 domains 38
1.4.1. Investigation of the replication of the IE1 deletion virus CR208 in PML-kd
cells 38
1.4.2. Analysis of HCMV-induced hDaxx degradation at immediate-early times
after infection 39
1.5. Differential growth complementation of various HCMV mutants in PML-
versus hDaxx-kd cells 41
1.5.1. The growth defect of a pp71-deleted HCMV is preferentially alleviated in
hDaxx-kd cells 42
1.5.2. The impaired growth phenotype of the IE1 deletion mutant CR208 is
especially abrogated after depletion of PML 43
1.5.3. Generation of a HCMV mutant with a deletion within the major IE
regulatory region 44
1.5.3.1. IE gene expression of AD169/del-MIEP is particularly stimulated in PML-
negative cells 47
1.6. Comparison of HCMV IE gene expression in siDaxx1+siPML2 double-kd
cells to that in the respective single-kd HFFs 48

2. Characterization of the spatial association of the viral regulatory proteins
IE2p86 and pp71 with the subnuclear structure ND10 49
2.1. Investigation of the determinants for IE2 subnuclear localization 50
2.1.1. Influence of ND10 domains on the subnuclear distribution of transiently
expressed IE2 51
2.1.2. Effect of ND10 structures on IE2 accumulations in HCMV-infected cells 52
2.1.2.1. Reorganization of ND10-like structures after HCMV infection of PML-kd
HFFs 52
2.1.2.2. IE1-induced dispersal of rearranged ND10-like accumulations in PML-kd
cells 54
2.1.3. Characterization of a recombinant HCMV expressing an EGFP-tagged
version of IE2 56
2.1.3.1. Growth analysis of the AD169/IE2-EGFP virus 56
2.1.3.2. Protein expression kinetics during AD169/IE2-EGFP lytic infection 57
2.1.3.3. Determination of the particle-to-PFU ratio of AD169/IE2-EGFP compared
to wild-type HCMV 59
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2.1.4. Generation of mCherry-Sp100-expressing HFFs for visualization of ND10
domains in live-infected cells 60
2.1.4.1. Differential localization of IE2 and ND10 in mCh-Sp100-expressing cells 61
2.1.4.2. Analysis of the dynamics of fluorescently labeled Sp100 and IE2 using
live cell microscopy 62
2.1.5. The number of IE2 foci is dependent on the input multiplicity of infection 63
2.1.6. Development of IE2 accumulations into viral replication compartments
during the course of infection 64
2.1.7. Association of IE2 with parental viral genomes at early stages of infection 65
2.1.8. Evaluation of the requirement of the DNA-binding activity of IE2 for its
subnuclear localization 66
2.2. Investigation of the determinants for pp71 subnuclear localization 67
2.2.1. Characterization of a recombinant HCMV coding for an EYFP-pp71 fusion
protein 68
2.2.1.1. Growth analysis of the AD169/EYFP-pp71 virus 68
2.2.1.2. Comparison of the protein expression kinetics between AD169/EYFP-pp71
and wild-type HCMV 69
2.2.1.3. Enhanced initiation of IE gene expression after low MOI infection with
AD169/EYFP-pp71 70
2.2.1.4. Dissection of the virus tegument composition of AD169/EYFP-pp71 72
2.2.1.5. HDaxx degradation is not significantly increased after AD169/EYFP-pp71
infection 73
2.2.1.6. Enhanced viral DNA replication after infection with AD169/EYFP-pp71 74
2.2.2. Analysis of pp71 subnuclear localization during infection with
AD169/EYFP-pp71 76
2.2.3. Examination of the intracellular distribution of pp71 in the absence of PML
or hDaxx 78

F. Discussion 81
G. Abreviatons 97
H. References 98
I. Apendix 114
Summary 1
A. Summary
Human cytomegalovirus (HCMV) infection commonly results in the association of viral
genomes and important regulatory proteins like pp71, IE1, or IE2 with a cellular subnuclear
structure known as the nuclear domain 10 (ND10). Since only ND10-associated genomes
initiate viral gene expression, ND10 domains were hypothesized