RNA-binding of the human cytomegalovirus transactivator protein UL69 and its role in mRNA export [Elektronische Ressource] / vorgelegt von Zsolt Toth

Zttoth

Sujets

• Biologie

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 41
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

RNA-binding of the human cytomegalovirus transactivator protein
UL69 and its role in mRNA export





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













vorgelegt von
Zsolt Toth
aus Szombathely




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


















Tag der mündlichen Prüfung: 09.10.2006.

Vorsitzender der Promotionskommission: Prof. Dr. Donat P. Häder

Erstberichterstatter: Prof. Dr. Wolfgang Hillen

Zweitberichterstatter: Prof. Dr. Michael Mach















The more you know, the less you understand the world.
Table of contents

A. SUMMARY 1

A. ZUSAMMENFASSUNG 2
B. INTRODUCTION 3
1. General features of human cytomegalovirus 3
2. Cellular mRNA export in higher eukaryotes 6
3. Viral mRNA export 9
3.1. Nuclear export of retrovirus mRNAs 9
3.2. Nuclear export of herpesvirus mRNAs 11
4. The UL69 protein of human cytomegalovirus 12
C. OBJECTIVES 14
D. MATERIALS AND METHODS 15
1. Biological materials 15
1.1. Bacteria 15
1.2. Eukaryotic cell cultures 15
1.3. Virus strains 15
1.4. Antibodies 16
1.4.1. Monoclonal antibodies 16
1.4.2. Hybridoma tissue culture supernatants 16
1.4.3. Polyclonal antibodies 16
1.4.4. Secondary antibodies 17
1.5. Nucleic acids 17
1.5.1. Oligonucleotides 17
1.5.2. Vectors and vector series 19
1.5.3. Ready-to-use DNA constructs 20
1.5.4. New DNA constructs 21
2. Enzymes, chemicals and mediums 24
2.1. Enzymes 24
2.2. Media 24
2.3. Chemicals 25
3. Standard buffers 25
4. Standard molecular biology techniques 25
5. RNA techniques 26
5.1. Total cellular RNA preparation from transfected tissue culture cells 26
5.2. Cytoplasmic RNA preparation from transfected tissue culture cells 26
5.3. In vitro transcription 27
5.4. Ribonuclease protection assay 27
5.5. Reverse transcription polymerase chain reaction (RT-PCR) 28
5.6. RNA-protein interaction assays 28
5.6.1. RNA-immunoprecipitation 28
5.6.2. Northwestern blotting assay 29
5.6.3. RNA electrophoretic mobility shift assay (REMSA) 29
5.7. CAT mRNA export assay 29
6. Cell culture techniques 30
6.1. Maintenance of cell cultures 30
6.2. Transfection and infection 30
6.3. Indirect immunofluorescence analysis 31
7. Purification and analysis of proteins and protein-protein interactions 31
7.1. Purification of GST fusion proteins expressed in E. coli 31
7.2. Purification of Histidine-tagged fusion proteins expressed in E. coli 32
7.3. Purification of FLAG-fusion proteins expressed in mammalian cells 32
7.4. Co-immunoprecipitation 33
7.5. Coomassie blue and silver staining 33
8. Generation of a deletion mutant of HCMV (Bacmid technology) 33
8.1. Homologous recombination using linear DNA fragments 34
8.2. Preparation and restriction enzyme digestion of bacmid DNA 34
8.3. Southern blotting analysis with biotinylated DNA probes 34
8.4. Transfection of bacmid DNA into primary human fibroblasts 35
9. Virus titration based on IE1 gene expression 35
E. RESULTS 37
1. Examination of the effect of pUL69 on nuclear mRNA export 37
1.1. Assaying nuclear mRNA export by transient transfection experiments 37
1.2. Evidence for enhanced cytoplasmic accumulation of unspliced mRNA in the presence
of pUL69 38
1.3. Confirmation that pUL69 promotes the cytoplasmic accumulation of unspliced
CAT mRNA 40
2. Identification and characterization of the RNA-binding domain of pUL69 in vitro 42
Functional domains of the herpesviral mRNA export factors involved in nuclear RNA 2.1.
export 42
2.2. RNA-binding activity of prokaryotically expressed pUL69 in vitro 42
2.2.1. Mapping of the RNA binding domain of pUL69 44
2.2.2. Essential role of the arginine-rich clusters at the N-terminus of pUL69 for RNA
binding in vitro 45
2.2.3 . Loss of pUL69 RNA-binding by combined deletion of the arginine-rich clusters 47
2.3. Immunopurification of FLAG-tagged pUL69 from mammalian cells 49
2.4. RNA-binding activity of eukaryotically expressed, full-length pUL69 in vitro 50
2.5. Non-specific RNA-binding activity of pUL69 in vitro 51
3. Characterization of the RNA-binding deficient pUL69 mutants by transfection
analyses of mammalian cells 53
3.1. The subcellular localization of the in-frame deletion pUL69 mutants in mammalian cells 53
3.2. RNA export activity of pUL69 mutants in transfected cells 54
3.3. Nucleocytoplasmic shuttling activity of the RNA binding deficient pUL69 mutants 56
3.4. In vivo interaction of the RNA binding deficient pUL69 mutants with the cellular
proteins UAP56, URH49 and hSPT6 57
3.5. Investigation of the in vivo interaction of pUL69 with RNA in living cells 59
3.5.1. Association of pUL69 with unspliced CAT mRNA in vivo 59
3.5.2. Investigation of mRNA export by the pUL69 RNA binding domain with a
tethering assay 62
4. Construction and characterization of a UL69 deficient human cytomegalovirus
mutant 64
4.1. Deletion of the UL69 gene from HCMV AD169 by homologous recombination
using a linear DNA fragment 64
4.2. Confirmation of the deletion of UL69 by PCR, restriction enzyme digestion and
Southern blotting 65
4.3. Reconstitution and titration of a UL69 deficient human cytomegalovirus mutant 67
4.4. Confirmation of the deletion of UL69 by western blotting and immuno-fluorescence
analysis of infected cells 68
4.5. Comparison of the kinetics of viral protein expression between DUL69 and wildtype
viruses by western blot analyses 69
F. DISCUSSION 73
1. Common features of pUL69 with herpesviral mRNA export factors 73
2. Interaction of pUL69 with RNA 74
3. Role of the RNA-binding activity of herpesviral mRNA export factors for nuclear
mRNA export 76
4. Functions of pUL69 during HCMV replication 79
G. REFERENCES 83 1
A. Summary
The human cytomegalovirus protein UL69 is a member of a family of homologous
regulatory proteins within the Herpesviridae which includes the proteins ICP27 of
herpes simplex virus type 1 and EB2 of Epstein-Barr virus. Both ICP27 and EB2 have
been shown to promote the nuclear export of a set of viral mRNAs via binding to the
cellular mRNA export factor REF. Furthermore, a direct RNA-binding activity of
ICP27 and EB2 was found to be essential for their stimulating effects on viral mRNA
export. Recently, pUL69 has been demonstrated to share several properties with its
herpesviral homologues suggesting that pUL69 might function as a viral mRNA
export factor of human cytomegalovirus. It has been demonstrated that (i) pUL69
shuttles between the nucleus and the cytoplasm, (ii) the nucleocytoplasmic shuttling
activity is essential for pUL69-mediated activation of gene expression and (iii) pUL69
interacts with cellular proteins involved in mRNA export. However, a direct role of
pUL69 in mRNA export has not yet been shown. This study demonstrates that pUL69
can induce the cytoplasmic accumulation of an unspliced CAT reporter mRNA in
transfected cells suggesting that pUL69 functions as an RNA export factor. One of the
characteristic features of the viral mRNA export factors is the direct interaction with
their RNA targets. Several experiments were performed to demonstrate that pUL69
can interact with RNA both in vivo and in vitro via a complex N-terminal RNA-
binding domain consisting of three arginine-rich motifs. Interestingly, the RNA-
binding domain of pUL69 overlaps with both the NLS and the binding site of the
cellular mRNA export factors UAP56 and URH49. Whereas the deletion of the
UAP56/URH49-binding site abolished pUL69-mediated mRNA export in transfected
cells, an RNA-binding deficient pUL69 mutant which can still interact with
UAP56/URH49 retained its RNA export activity. This suggests that, in contrast to its
homologues, direct RNA interaction is not a prerequisite for the RNA export activity
of pUL69, leastwise in a transient transfection system. To address the in vivo role of
pUL69 for the HCMV replication cycle, a UL69 deficient HCMV ( DUL69) was
generated. The analysis of expression of representative immediate early, early and late
HCMV proteins in DUL69-infected cells revealed that pUL69 appears to be required
for the efficient expression of a subset of early and several late HCMV genes. The
RNA transcripts of these genes could represent targets for pUL69-mediated mRNA
export. 2
A. Zusammenfassung
Das Protein pUL69 des humanen Cytomega