Genetic analysis of the mouse cytomegalovirus nuclear egress complex [Elektronische Ressource] / vorgelegt von Mirela Popa

Popa, Ouveau Théâtre" : Eugène Ionesco Samuel Beckett Arthur Adamov Alenation And Absurd In The "New Theatre" : Eugène Ionesco Samuel Beckett Arthur Adamov" Mirela

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Genetic analysis of the mouse
cytomegalovirus nuclear egress complex







Dissertation der Fakultät für Biologie
der Ludwig-Maximilians-Universität München
zur Erlangung des Dr. rer. nat.



vorgelegt von
Mirela Popa
München, June 2009








































Dissertation eingereicht am: 23.06.2009

Erstgutachterin: Prof. Dr. Bettina Kempkes
Zweitgutachterin: Prof. Dr. Elisabeth Weiß
Sondergutachter: Prof. Dr. Ulrich Koszinowski

Tag der mündlichen Prüfung: 16.02.2010



Ehrenwörtliche Versicherung

Hiermit versichere ich, dass ich die vorliegende Arbeit selbstständig
angefertigt habe. Es wurden keine anderen als die angegebenen
Hilfsmittel und Quellen verwendet. Ich habe weder anderweitig versucht
eine Dissertation einzureichen oder eine Doktorprüfung durchzuführen,
noch habe ich diese Dissertation oder Teile derselben einer anderen
Prüfungskommission vorgelegt.


München,23.06.2009

________Mirela Popa_______



Table of content
Table of content

Chapter 1 Summary………………………………………………………………..1
Chapter 2 Introduction……………………………………………………………..2
Chapter 2.1 Herpesviridae: a general introduction………………………………...2
Chapter 2.1.1 The structure of herpesvirus particles………………………………..4
Chapter 2.1.2 Lytic replication of herpesvirus………………………………………..6
Chapter 2.1.2.1 Viral entry………………………………………………………………..6
Chapter 2.1.2.2 Kinetics of viral gene expression……………………………………...8
Chapter 2.1.2.3 Replication and gene expression……………………………………..9
Chapter 2.1.2.4 Herpesvirus assembly and egress…………………………………..11
Chapter 2.1.2.4.1 Nuclear events in herpesviruses morphogenesis………………….11
Chapter 2.1.2.4.2 Cytoplasmic maturation of herpesvirus particles…………………..16
Chapter 2.2 Dominant negative mutants of cellular and viral proteins…………18
Chapter 2.3 Aims and concepts……………………………………………………20
Chapter 3 Materials………………………………………………………………..23
Chapter 3.1 Devices…………………………………………………………………23
Chapter 3.2 Consumables
Chapter 3.3 Kits and reagents……………………………………………………...24
Chapter 3.4 Buffers and solutions………………………………………………….26
Chapter 3.5 Media…………………………………………………………………...28
Chapter 3.6 Bacteria and cell lines………………………………………………...28
Chapter 3.7 DNA……………………………………………………………………..29
Chapter 3.7.1 BACs……………………………………………………………………29
Chapter 3.7.2 Plasmids………………………………………………………………..30
Chapter 3.8 Viruses………………………………………………………………….30
I Table of content
Chapter 4 Methods………………………………………………………………...32
Chapter 4.1 PCRs……………………………………………………………………32
Chapter 4.2 Cloning steps…………………………………………………………..33
Chapter 4.3 Propagation of electro-competent Escherichia coli cells………....37
Chapter 4.4 Electro-transformation of Escherichia coli strains…………….…...37
Chapter 4.5 Flp recombination....…………………………………………………. 38
Chapter 4.6 Preparation of glycerol stocks for Escherichia coli cells.………….39
Chapter 4.7 Low copy plasmids mini preps……………………………………….39
Chapter 4.8 Low copy plasmids maxi preps………………………………………40
Chapter 4.9 Screening for M53 single insertion mutants at the FRT site of wt
MCMV BAC……………………………………………………………40
Chapter 4.10 Transfection of MEFs………………………………………………...41
Chapter 4.11 Infection of M2-10B4 cells…………………………………………...42
Chapter 4.12 Preparation of virus inoculums………………………………………43
Chapter 4.13 Semi-quantitative conditional expression…………………………..44
Chapter 4.14 Virus titration - on MEFs (inoculum and stock)…………………….44
Chapter 4.15 Viral growth kinetics ……………………..…………………………...45
Chapter 4.16 Preparation of virus stocks…………………………………………...46
Chapter 4.17 293 cells transfection………………………………………………….47
Chapter 4.18 Infection of NIH3T3 cells……………………………………………..48
Chapter 4.19 SDS-PAGE……………………………………………………………..49
Chapter 4.20 Western-blot analysis………………………………………………….49
Chapter 4.21 HA-tag pulldown….…………………………………………………….50
Chapter 4.22 Southern-blot analysis…………………………………………………51
Chapter 4.23 Confocal microscopy and indirect immunofluorescent staining….53
Chapter 5 Results………………………………………………………………….55
II Table of content
Chapter 5.1 Genetic screen for inhibitory M53 mutants…...…………………….55
Chapter 5.2 Conditional expression of inhibitory M53 mutants…………………59
Chapter 5.3 The stability of the inhibitory effect in different cell types…………62
Chapter 5.4 The stability of the inhibitory phenotype at high virus load……….64
Chapter 5.5 Synthesis of the inhibitory protein…………………………………...65
Chapter 5.6 EM analysis of MCMV recombinant expressing the DN s309……67
Chapter 5.7 Tagging of the M53 ORF …………………………………………….68
Chapter 5.7.1 Flag-tagged M53 constructs hold the inhibitory feature…………..68
Chapter 5.7.2 Conditional expression of Flag-tagged constructs by MCMV
recombinants………………………………………………….……….70
Chapter 5.8 Subcellular localization of NEC members…………………………..73
Chapter 5.9 Analysis of the DN s309 phenotype..............................................77
Chapter 5.10 s309R effects on cleavage/packaging are independent of NEC
formation……………………………………………………………….81
Chapter 6. Discussion……………………………………………………………...86
Chapter 6.1 The MCMV M53 C-terminal region harbours at least two functional
domains………………………………………………………………...86
Chapter 6.2 M53 CR4 functional domain and MCMV nucleocapsids egress
defect …………………………………………………………………..90
Chapter 6.3 M53 CR4 mutants induce defects in capsid maturation…………..93
Chapter 6.4 The effect of the DN CR4 mutation on nucleocapsid maturation is
independent of NEC formation.……………………………………...97
Chapter 6.5 The MCMV M53 screen and further screens………………………98
Chapter 7 Conclusions…………………………………………………………..100
Chapter 8 Outlook………………………………………………………………..101
Reference list……………………………………………………………………………...103
III Table of content
Supplementary information………………………………………………………………116
S.1 List of abbreviations………………………………………………………………….116
S.2 List of figures………………………………………………………………………….121
S.3 List of tables…………………………………………………………………………..122
S.4 Posters and oral presentations……………………………………………………..123
S.5 Publications…………………………………………………………………………...123
S.6 Acknowledgements…………………………………………………………………..124
S.7 Curriculum vitae………………………………………………………………………125

IV Chapter 1 Summary
1. Summary

Cytomegaloviruses represent a part of the beta herpesviruses subfamily
characterized by a restricted host range and a slow replication cycle. DNA
replication, late viral transcription, and viral nucleocapsid maturation occur in the
replication compartments (RCs) within the nuclei of infected cells. During infection,
RCs expand from small replication sites to large domains, disrupting the nuclear
interior by compressing and marginalizing host chromatin which represents a natural
barrier for the export of virus nucleocapsids to the cytoplasm. Two conserved
essential murine cytomegalovirus (MCMV) proteins, M50 and M53, play a critical role
in capsid export from nucleus to the cytosol where the final events of the virus
maturation take place.
To understand more about the function of the M53 protein, we have analyzed
a comprehensive set of loss-of-function mutants derived from a random genetic
screen to identify the dominant negative (DN) alleles of the M53 gene. Mutations with
inhibitory effect have accumulated within two conserved regions of M53.
Recombinant MCMVs were constructed for conditional expression of these inhibitory
mutants in order to analyze the phenotype they induced. Conditional expression of
single inhibitory M53 mutants down-regulated virus replication up to one million folds.
Studies on this DN phenotype revealed a complete block of nuclear egress and,
unexpectedly, also an inhibition of viral capsids maturation.
This work proved that random mutagenesis followed by cis-complementation
based genetic screens is a valuable strategy to identify viral DN mutants and
provided the first evidence that DNA cleavage/packaging and capsid export are
coupled during cytomegalovirus infection identifying a new function for egress protein
M53.
1 Chapter 2 Introduction
2. Introduction

2.1 Herpesviridae: a general introduction
Herpesviruses are large enveloped viruses with linear double stranded