The role of Epstein-Barr virus nuclear antigen 3C in the immortalisation process of primary human B-lymphocytes [Elektronische Ressource] / von Madelaine Löfqvist

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• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 3
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

The role of Epstein-Barr virus nuclear antigen 3C in the
immortalisation process of human primary B-lymphocytes



Dissertation der Fakultät für Biologie der
Ludwig-Maximilians-Universität München
von
Madelaine Löfqvist
München, Februar 2004 First examiner: Prof. Dr. D. Eick
Second examiner: Prof. Dr. J. Parsch

Date of exam: 8 Juli 2004 Table of contents
1. Introduction........................................................................................................... 6
1.1 Herpesviruses...............................................................................................................6
1.1.1 The Epstein-Barr virus (EBV) ................................................................................................ 7
1.1.2 Malignancies associated with EBV........................................................................................ 8
1.1.3 EBV genetics using BACs ..................................................................................................... 8
1.2 In vitro infection of primary human B-lymphocytes as a model system for EBV
induced B-cell immortalisation .......................................................................................11
1.2.1 Formation of lymphoblastoid cell lines................................................................................. 11
1.2.2 Viral proteins involved in B-cell immortalisation .................................................................. 11
1.3 The Epstein-Barr nuclear antigen 3C (EBNA 3C) and its interaction with other
proteins .............................................................................................................................13
1.4 Aim of the project: Investigation of the importance of EBNA 3C in B-cell
immortalisation in the context of an EBV infection ......................................................16
2. Materials.............................................................................................................. 17
2.1 Antibodies...................................................................................................................17
2.2 Bacteria .......................................................................................................................17
2.3 Plasmids...18
2.4 Cells and Cell lines.....................................................................................................22
2.5 Oligonucleotides ........................................................................................................23
2.6 Reagents..24
3. Methods............................................................................................................... 26
3.1 Isolation and purification of nucleic acids...............................................................26
3.3 DNA analysis ..............................................................................................................30
3.4 Polymerase chain reaction (PCR).............................................................................32
3.5 Mutagenesis using Maxi-EBV plasmids...................................................................33
3.6 Cell culture and analysis of cells..............................................................................35
3.6.1 Cell culture conditions.......................................................................................................... 35
3.6.2 Establishment of HEK293 stable cell lines carrying Maxi-EBV plasmid.............................. 36
3.6.3 Production of infectious virus particles and titer determination ........................................... 36 3.6.4 Preparation of primary B-lymphocytes ................................................................................ 37
3.6.5 Infection of primary B-lymphocytes with EBV mutants and determination of the
immortalisation frequency............................................................................................................. 37
3.7 Immunofluorescence .................................................................................................38
3.8 Retrovirus production and concentration................................................................38
3.9 Protein analysis..........................................................................................................39
4. Results................................................................................................................. 42
4.1 Establishment of recombinant Epstein-Barr viruses..............................................42
4.2 Generation of nine recombinant Maxi-EBV genomes with partial deletions in
EBNA 3C............................................................................................................................44
4.2.1 Cloning of the recombination plasmids................................................................................ 44
4.2.2 Red αβγ mediated mutagenesis of EBNA 3C in the Maxi-EBV........................................... 47
4.2.3 Establishment of twelve producer cell lines and generation of virus stocks........................ 50
4.3 Infection of primary human B-lymphocytes with recombinant EBNA 3C EBVs ..52
4.3.1 Reduced immortalisation efficiency with EBNA 3C mutants compared to wild-type EBV... 52
4.3.2 Establishment of mutant EBNA 3C LCL clones................................................................... 56
4.3.2.1 Proliferation phenotype of LCLs carrying the different EBV mutants with deletions in
EBNA 3C .................................................................................................................................. 61
4.3.3 Expression of EBNA 3C in the established LCLs carrying mutant EBNA 3C ..................... 62
4.3.4 EBNA 3C deletion mutants alter the expression of EBNA 1, EBNA 2 and LMP 1.............. 63
4.3.5 EBNA 3C knock-out mutant EBV does not yield LCLs........................................................ 65
4.4 Generation of an inducible system for the investigation of the importance of
EBNA 3C in the initiation or maintenance phase of B-cell immortalisation ...............68
4.4.1 Generation of an inducible EBNA 3C knock-out system ..................................................... 68
4.4.1.1 The Cre/loxP system .................................................................................................... 68
4.4.1.2 The conditional Lox P flanked EBNA 3C Maxi-EBV mutant ........................................ 69
4.4.1.3 Expression of the Cre protein in LCLs using a retroviral vector................................... 71
4.4.1.4 Infection of B-cells with recombinant retrovirus............................................................ 72
4.4.1.5 Confirmation of Cre expression and EBNA 3C deletion............................................... 73
5. Discussion .......................................................................................................... 78
5.1 The role of EBNA 3C in the immortalisation process of B-lymphocytes ..............79
5.1.1 EBNA 3C deletion mutants alter the expression of EBNA 1, EBNA 2 and LMP 1 84
5.2 The EBNA 3C knock-out phenotype.........................................................................85
5.3 A conditional EBNA 3C system.................................................................................86
6. Summary ............................................................................................................. 89 7. Abbreviations...................................................................................................... 91
8. Literature............................................................................................................. 93 Introduction 6
1. Introduction
1.1 Herpesviruses
The architecture of the virion is the criteria by which the members of the
Herpesviridae family are classified. All herpesviruses consist of a core containing a
large double stranded DNA, an icosadeltahedral capsid, tegument, and an envelope
containing viral glycoproteins on the surface. So far, nine herpesviruses have been
isolated from humans (HSV1, HSV2, HCMV, VZV, EBV, HHV 6A, HHV 6B, HHV 7
and HHV 8) (Kieff and Rickinson, 2001). Four biological properties are shared by the
herpesviruses. (i) They all express a number of proteins involved in nucleic acid
metabolism and processing of proteins (although the number of these enzymes may
vary from one herpesvirus to another). (ii) The synthesis of viral DNAs and the
assembly of the capsid take place in the nucleus. It is still unclear, however whether
herpesvirions undergo internal cellular maturation or how they obtain their
membraneous envelope (Enquist et al., 1998). (iii) All herpesviruses are able to
remain in their natural host in an inactive state called latency. (iv) Production of
infectious progeny virus is accompanied by the destruction of the infected cell.
Based on biological properties the herpesvirus family is been divided into three
subfamilies, the alpha-, beta- and gamma-herpesviruses. Members of the alpha-
herpesvirus subfamily are classified based on their variable host range, their relative
short reproduction cycle, efficient spread to cells, and their capacity to establish latent
infection primarily in sensory ganglia. Family members are human herpes simplex
virus 1 and 2 (HSV 1, HSV 2), as well as Var