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Investigation of murine cytomegalovirus US22 gene family members m139 and m142 [Elektronische Ressource] / vorgelegt von Karina Holak

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Max-von-Pettenkofer Institut für Medizinische Mikrobiologie und Hygiene Abteilung Virologie der Ludwig-Maximilians-Universität München Vorstand: Prof. Dr. med. Ulrich Koszinowski Investigation of murine cytomegalovirus US22 gene family members m139 and m142 Dissertation zum Erwerb des Doktorgrades der Medizin der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Karina Holak aus Krakau 2007 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Prof. Dr. med. Ulrich Koszinowski Mitberichterstatter: Prof. Dr. H.G. Klobeck Prof. Dr. J. Bogner Mitbetreuung durch: Dr. rer. nat. Markus Wagner Dr. rer. nat. Carine Ménard Dekan: Prof. Dr. med. Dietrich Reinhardt Tag der mündlichen Prüfung: 28.06.2007 To my parents. Table of contents I A. INTRODUCTION ...................................................................................................... 1 1 Cytomegalovirus as a herpesvirus ............................................................................. 1 1.1 The group of herpesviruses ................................................................................... 1 1.2 Cytomegalovirus .......................................................................................

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Published 01 January 2007
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Max-von-Pettenkofer Institut für Medizinische Mikrobiologie und Hygiene
Abteilung Virologie der Ludwig-Maximilians-Universität München
Vorstand: Prof. Dr. med. Ulrich Koszinowski








Investigation of murine cytomegalovirus US22
gene family members m139 and m142








Dissertation zum Erwerb des Doktorgrades der Medizin
der Medizinischen Fakultät der Ludwig-Maximilians-Universität München


















vorgelegt von

Karina Holak
aus Krakau

2007 Mit Genehmigung der Medizinischen Fakultät der Universität München



























Berichterstatter: Prof. Dr. med. Ulrich Koszinowski



Mitberichterstatter: Prof. Dr. H.G. Klobeck
Prof. Dr. J. Bogner





Mitbetreuung durch: Dr. rer. nat. Markus Wagner
Dr. rer. nat. Carine Ménard

Dekan: Prof. Dr. med. Dietrich Reinhardt


Tag der mündlichen Prüfung: 28.06.2007





















To my parents. Table of contents I
A. INTRODUCTION ...................................................................................................... 1
1 Cytomegalovirus as a herpesvirus ............................................................................. 1
1.1 The group of herpesviruses ................................................................................... 1
1.2 Cytomegalovirus .................................................................................................... 2
1.2.1 Structure of the cytomegalovirus virion ............................................................ 2
1.2.2 Organization of the cytomegalovirus genome ................................................... 3
1.2.3 Gene expression and replication of CMV ......................................................... 5
1.2.4 Pathogenesis of cytomegalovirus infection: mechanisms of dissemination
and tissue tropism .............................................................................................. 6
1.2.5 Immune response and latency in cytomegalovirus infection ............................ 6
2 Clinical aspects of cytomegalovirus infection ........................................................... 8
2.1 Epidemiology and transmission of cytomegalovirus ............................................ 8
2.2 Clinical manifestations of cytomegalovirus ........................................................ 10
2.3 Diagnosis of cytomegalovirus infection .............................................................. 11
2.4 Therapy of cytome................................................................. 12
3 Generation of recombinant cytomegalovirus .......................................................... 13
3.1 Methods of mutagenesis used before bacterial artificial chromosome
technology ........................................................................................................... 13
3.1.1 Chemical mutagenesis in infected cells ...........................................................
3.1.2 Site-directed mutagenesis by homologous recombination in CMV-
infected cells .................................................................................................... 13
3.1.3 Regenerating virus from overlapping cosmid clones ...................................... 15
3.2 CMV mutagenesis with bacterial artificial chromosomes in E. coli ................... 15
3.2.1 Cloning herpesvirus genomes as bacterial artificial chromosomes ................. 15
3.2.2 Random mutagenesis of cytomegalovirus BACs with transposons ................ 16
3.2.3 Allelic exchange using linear DNA fragments ................................................ 18
4 Murine cytomegalovirus as a model for human cytomegalovirus infection ........... 20
4.1 egalovirus infection ...................................................................... 20
4.2 Function of open reading frames in human and murine cytomegalovirus .......... 20
4.3 Gene families in human and murine cytomegalovirus ........................................ 21
5 Goal of this thesis .................................................................................................... 23
Table of contents II
5.1 Generation of US 22 gene homolog mutants m139, m140, and m142
MCMV ................................................................................................................ 23
5.2 Characterization of MCMV mutants m139, m140 and m142 ............................. 24

B. MATERIALS AND METHODS ............................................................................. 25
1 Materials .................................................................................................................. 25
1.1 Reagents .............................................................................................................. 25
1.2 Bacteria ................................................................................................................ 27
1.3 Plasmids ............................................................................................................... 27
1.4 Antibodies ............................................................................................................ 29
1.5 Cells and viruses .................................................................................................. 30
1.6 Oligonucleotides 30
2 Methods ................................................................................................................... 32
2.1 Isolation und purification of nucleic acids .......................................................... 32
2.1.1 Analytical isolation of plasmid DNA from bacteria (mini preparation
of plasmid DNA) ............................................................................................. 32
2.1.2 Quantitative isolation of plasmid DNA from bacteria (maxi preparation
of plasmid DNA) 33
2.1.3 Analytical isolation of BAC DNA from bacteria (mini preparation
of plasmid DNA) 33
2.1.4 Quantitative isolation of BAC DNA from bacteria (maxi and midi
preparation of BAC DNA) .............................................................................. 34
2.1.5 Concentration of DNA by ethanol precipitation ............................................. 35
2.1.6 Determination of DNA concentration ............................................................. 35
2.2 Cloning of DNA .................................................................................................. 35
2.2.1 Digestion of DNA with restriction enzymes ................................................... 35
2.2.2 Dephosphorylation of DNA ............................................................................ 35
2.2.3 Treatment of DNA with Klenow polymerase.................................................. 36
2.2.4 Purification of DNA fragments from agarose gels .......................................... 36
2.2.5 Ligation of DNA .............................................................................................. 36
2.2.6 Preparation of electro-competent bacteria ....................................................... 36
2.2.7 Preparation of chemical-competent bacteria ................................................... 37
Table of contents III
2.2.8 Electroporation of bacteria .............................................................................. 37
2.2.9 Chemical transformation of bacteria ............................................................... 38
2.2.10 Preservation of bacteria ................................................................................... 38
2.3 Analysis of DNA ................................................................................................. 38
2.3.1 Agarose gel electrophoresis ............................................................................. 38
2.3.2 Sequencing of DNA ........................................................................................ 39
2.4 Polymerase-chain reaction (PCR) ....................................................................... 39
2.5 Mutagenesis of MCMV BAC plasmids .............................................................. 40
2.5.1 Generation of PCR fragments with viral homologies ..................................... 40
2.5.2 Purification of PCR fragments ........................................................................ 40
2.5.3 Generation of electro-competent and arabinose-induced DH10B bacteria ..... 41
2.5.4 Transformation of the PCR fragment into the arabinose-induced bacteria ..... 41
2.5.5 Deletion of the FRT-flanked selection marker ................................................ 41
2.5.6 Viral reconstitution of MCMV BAC plasmids ............................................... 43
2.6 Proteins in infected cells ...................................................................................... 43
2.6.1 SDS-PAGE gel electrophoresis ....................................................................... 43
2.6.2 Western blotting .............................................................................................. 44
2.7 Cell culture .......................................................................................................... 45
2.7.1 Thawing cells ................................................................................................... 45
2.7.2 Freezing cells 46
2.7.3 Transfection of eukaryotic cells with calcium-phosphate precipitation
and glycerol shock ........................................................................................... 46
2.7.4 ith Superfect transfection reagent ............. 47
2.8 Generation of MCMV viral stock ........................................................................ 47
2.9 Viral titration of MCMV with standard plaque assay ......................................... 48
2.10 Viral titration of MCMV with the TCID (median tissue culture infectious 50
dose) method ....................................................................................................... 49
2.11 Isolation of mouse embryonic fibroblasts from the mouse .................................

C. RESULTS .................................................................................................................. 50
1 Mutants ΔATG-m139 and ΔATG-m142 were generated by site-directed
mutagenesis ............................................................................................................ 50
Table of contents IV
1.1 PCR fragments with flanking viral homologies to ORF m139, m140 and
m142 were generated ........................................................................................... 50
1.2 Site-directed mutants of ORF m139 and m142 were generated .......................... 52
1.3 The kanamycin cassette was excised from mutant ΔATG-m139+kan and mutant
ΔATG-m142+kan ................................................................................................ 54
1.4 Site-directed mutagenesis of ORF m139 and m142 introduces a minimal
genetic alteration .................................................................................................. 57
2 Mutant ΔATG-m139 has wild-type properties in NIH3T3 fibroblasts, but
shows attenuated growth in IC-21 macrophages .................................................... 58
2.1 Growth kinetics in NIH3T3 fibroblasts ............................................................... 58
2.2 Growth kinetics in IC-21 macrophages 59
3 The gene products of gene m139 interact with the gene products of genes
m140 and m141 ...................................................................................................... 62
3.1 The complex translational region of m139 to m143 ........................................... 62
3.2 Mutant ΔATG-m139 reduces the presence of the m140 and m141 proteins ...... 63
3.3 ΔATG-m139 expresses a truncated protein of m139 .......................................... 64
3.4 Gene products of m139, m140, and m141 interact on the protein level.............. 65
4 Gene m142 is essential for MCMV replication ....................................................... 65
4.1 Is ORF m142 essential for viral growth? ............................................................ 65
4.2 Construction of site-directed MCMV mutants of ORF m142 ............................. 66
4.3 ORF m142 has a lethal phenotype ....................................................................... 68
4.3.1 Rescuing essential genes ................................................................................. 68
4.3.2 Mutant ΔATG-m142 and ΔATG-m142/FRT did not reconstitute viral
progeny after transfection ................................................................................ 69
4.3.3 Fragment HindIII-I rescued ΔATG-m142/GFP .............................................. 70
4.3.4 The ectopic gene m142 rescued the ΔATG-m142/GFP mutant ...................... 70

D. DISCUSSION ............................................................................................................ 74
1 Targeted and random mutagenesis strategies of BAC cloned herpesvirus
genomes .................................................................................................................. 74
2 Analysis of the MCMV US22 gene family homologs m139 and m142 ................. 77
Table of contents V
2.1 Mutant ΔATG-m139 has a macrophage specific growth defect ......................... 77
2.2 Gene products of m139, m140, and m141 interact.............................................. 80
2.3 Gene m142 is essential ........................................................................................ 81

E. SUMMARY (English and German version) .............................................................. 84

F. REFERENCE LIST .................................................................................................. 88

G. ABBREVIATIONS ................................................................................................ 108

H. APPENDIX ............................................................................................................. 110
Danksagung
Publikation
Curriculum vitae
Erklärung
Figures and Tables VI
FIGURES AND TABLES

A. Introduction
Fig. A 1 The CMV virion ................................................................................................ 2
Fig. A 2 CMV genome structure. .................................................................................... 4
Fig. A 3 Characteristics of cytomegalovirus infection in pregnancy .............................. 9
Fig. A 4 Congenital HMCV infection ........................................................................... 10
Fig. A 5 Methods of herpesvirus mutagenesis before BAC technology ....................... 14
Fig. A 6 Random transposon mutagenesis of MCMV BAC plasmids .......................... 17
Fig. A 7 MCMV BAC mutagenesis. ............................................................................. 19
Tab. A 1 US 22 gene family homologs in MCMV and HCMV .................................... 22

B. Materials and Methods
Tab. B 1 Sequences of oligonucleotides ........................................................................ 31
Tab. B 2 Conditions for PCR ......................................................................................... 39
Fig. B 1 Efficacy of the excision of the kanamycin cassette ........................................ 42

C. Results
Fig. C 1 Generation of linear PCR fragments ............................................................... 51
Fig. C 2 Linear PCR fragments ..................................................................................... 52
Fig. C 3 Plasmid pBAD αβγ .......................................................................................... 52
Tab. C 1 Overview of the generated MCMV BAC plasmid mutants ............................ 53
Fig. C 4 Mutant ΔATG-m139 was generated by site-directed mutagenesis of ORF
m139................................................................................................................ 55
Fig. C 5 Mutant ΔATG-m142 was generated by site-directed mu
m142................................................................................................................ 56
Fig. C 6 Traces of mutagenesis ..................................................................................... 57
Tab. C 2 Mean viral titers of wild-type MCMV (MW 97.01, Tn-m152), mutant
ΔATG-m139, and transposon mutant Tn-m139 at day 0, 1, 4 and 6 postin-
fection in NIH 3T3 fibroblasts ........................................................................ 58
Fig. C 7 In vitro growth of mutant ΔATG-m139 in NIH3T3 fibroblasts ..................... 59 Figures and Tables VII
Tab. C 3 Mean viral titers of wild-type MCMV (MW 97.01, Tn-m152), mutant
ΔATG-m139 and transposon mutant Tn-m139 at day 0, 1, and 6 postinfec-
tion in IC-21 macrophages .............................................................................. 60
Fig. C 8 In vitro growth of mutant ΔATG-m139 in IC-21 macrophages ..................... 61
Fig. C 9 Transcript map of genes m139 to m143 .......................................................... 62
Fig. C 10 Western blot analysis with polyclonal antisera against protein m140 ............ 63
Fig. C 11 We141 ............ 64
Fig. C 12 We139 ............ 65
Fig. C 13 Transcript map of genes m142 to m143 66
Fig. C 14 Mutant ΔATG-m142/FRT was generated by site-directed mutagenesis of
plasmid pSM3fr/GFP ...................................................................................... 67
Fig. C 15 Mutants of essential genes .............................................................................. 69
Tab. C 4 Plaque formation on NIH3T3 cells after transfection of the MCMV BAC
plasmids .......................................................................................................... 70
Fig. C 16 Complementation of the ΔATG-m142 genome by insertion of the m142
gene at an ectopic position .............................................................................. 72
Fig. C 17 Construction of mutant ΔATG-m142/m142E ................................................. 73