«Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München von Madelaine Löfqvist München, Februar 2004 First examiner: ...»
The role of Epstein-Barr virus nuclear antigen 3C in the
immortalisation process of human primary B-lymphocytes
Dissertation der Fakultät für Biologie der
München, Februar 2004
First examiner: Prof. Dr. D. Eick
Second examiner: Prof. Dr. J. Parsch
8 Juli 2004
Date of exam:
Table of contents
1.1.1 The Epstein-Barr virus (EBV)
1.1.2 Malignancies associated with EBV
1.1.3 EBV genetics using BACs
1.2 In vitro infection of primary human B-lymphocytes as a model system for EBV induced B-cell immortalisation
1.2.1 Formation of lymphoblastoid cell lines
1.2.2 Viral proteins involved in B-cell immortalisation
1.3 The Epstein-Barr nuclear antigen 3C (EBNA 3C) and its interaction with other proteins
1.4 Aim of the project: Investigation of the importance of EBNA 3C in B-cell immortalisation in the context of an EBV infection
2.4 Cells and Cell lines
3.1 Isolation and purification of nucleic acids
3.3 DNA analysis
3.4 Polymerase chain reaction (PCR)
3.5 Mutagenesis using Maxi-EBV plasmids
3.6 Cell culture and analysis of cells
3.6.1 Cell culture conditions
3.6.2 Establishment of HEK293 stable cell lines carrying Maxi-EBV plasmid
3.6.3 Production of infectious virus particles and titer determination
3.6.4 Preparation of primary B-lymphocytes
3.6.5 Infection of primary B-lymphocytes with EBV mutants and determination of the immortalisation frequency
3.8 Retrovirus production and concentration
3.9 Protein analysis
4.1 Establishment of recombinant Epstein-Barr viruses
4.2 Generation of nine recombinant Maxi-EBV genomes with partial deletions in EBNA 3C
4.2.1 Cloning of the recombination plasmids
4.2.2 Red αβγ mediated mutagenesis of EBNA 3C in the Maxi-EBV
4.2.3 Establishment of twelve producer cell lines and generation of virus stocks
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
188.8.131.52 Proliferation phenotype of LCLs carrying the different EBV mutants with deletions in EBNA 3C
4.3.3 Expression of EBNA 3C in the established LCLs carrying mutant EBNA 3C
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
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
184.108.40.206 The Cre/loxP system
220.127.116.11 The conditional Lox P flanked EBNA 3C Maxi-EBV mutant
18.104.22.168 Expression of the Cre protein in LCLs using a retroviral vector
22.214.171.124 Infection of B-cells with recombinant retrovirus
126.96.36.199 Confirmation of Cre expression and EBNA 3C deletion
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
5.3 A conditional EBNA 3C system
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 alphaherpesvirus 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 Varicella Zoster virus (VZV). Classification of the beta-herpesvirus subfamily is based on their restricted host range, long reproduction cycle, and slow infection in culture. These viruses can establish latency in secretory glands, lymphoreticular cells, kidneys, and other tissue. Members of the beta-herpesvirus family are human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), and human herpes virus 6 and 7 (HHV 6, HHV 7). The gamma-herpesvirus are usually specific for either T or B-lymphocytes. All members replicate in vitro in lymphoblastoid cells, although some can cause lytic infection in epithelia cells and fibroblasts. Family members are Kaposi's sarcoma virus (KSV),
1.1.1 The Epstein-Barr virus (EBV) The acute stage of EBV infection normally takes place in childhood without major clinical symptoms. The primary infection is characterised by lytic DNA replication, expression of almost all viral genes, virus production, and lysis of the infected cell.
The host range for efficient EBV infection in vitro is restricted to primary Blymphocytes (Henle et al., 1967; Pope et al., 1968). After the first acute phase of infection latency is established and several copies of the 172 kbp EBV genome is maintained in B-cells as an episome. EBV can also establish latent infection in other cell types, including T- or natural killer (NK) cells, although the efficiency is low.
Latently infected primary B-lymphocytes become immortalized and yield growthtransformed lymphoblastoid cell lines (LCLs) (Henderson et al., 1977; Sugden and Mark, 1977). In this latent state, termed latency III, eleven of the approximately 90 genes of EBV are found to be expressed and induce an immune response in the host. B-cells, which present viral antigens, are eliminated and an EBV specific immunological memory develops. In some B-cells, probably memory B-cells, EBV substantially reduces the viral gene expression pattern to that of latency I or II. In latency I, only EBNA 1 is expressed, in contrast to latency II in which EBNA 1, LMP 1, LMP 2A as well as LMP 2B are expressed. As a consequence of switching to the latency state, the cell changes it´s phenotype indicated by altered surface marker expression and an arrest of proliferation. In latency I or II, EBV residing in B-cells evades the immune system and allows a persistent infection of the host. The frequency of EBV infected B-cells in the peripheral blood is 1 to 30 cells per 5x106 Bcells. After receiving an appropriate stimulus virus can be reactivated from latency (Rowe, 1999). The cells containing reactivated virus are normally eliminated by the immune system. In immunocompromised persons, for example patients suffering from AIDS or undergoing organ transplantation, EBV can be responsible for the development of B-cell tumours.
1.1.2 Malignancies associated with EBV
Several malignancies are associated with EBV. Among these are Hodgkin's disease, Burkitt´s lymphoma, and Nasopharyngeal carcinoma. Despite the fact that all EBV positive individuals possess latently infected B-cells in their blood, they rarely develop tumours since the immune system prevents uncontrolled proliferation of infected Bcells in immunological healthy persons. Several factors contribute to the pathogenesis of monoclonal Burkitt´s lymphoma. One is malaria infection, which suppresses a T-cell response and stimulates B-cell proliferation. Other unknown factors can also contribute to chromosomal translocations of the c-myc gene into immunoglobulin encoded regions of different chromosomes, leading to constitutive active c-myc expression.
Nasopharyngeal carcinomas are nose and throat epithelial cell tumours, which are especially frequent in China, indicating that genetic or environmental components may play a role. Hodgkin's lymphoma is the most common malignant lymphoma in the Western world. It is characterised by an altered lymph node structure and the presence of mononuclear Hodgkin- and Reed-Sternberg cells. Reed-Sternberg cells are transformed B-cells that are less differentiated but very malignant. Typically more than 98% of the tumour mass in Hodgkin´s lymphoma consists of non malignant tumour-invading T-cells. The lymphomas are most frequently seen in immunocompromised persons and the expression pattern of viral genes among different tumours varies.
Lymphoblastic B-cell lymphomas are characterized by the expression of eleven viral genes, which is typical for latency III. Nasopharyngeal carcinoma, Hodgkin- and Tcell lymphomas all express EBNA 1 (EBV nuclear antigen 1), LMP 1, LMP 2A and B, which characterises latency II. In Burkitt´s lymphoma only EBNA 1 and two small non-coding RNAs are expressed, indicating latency I.
1.1.3 EBV genetics using BACs
in which a mutation is directed to the gene of interest and the resulting phenotype is investigated, and (ii) forward genetics in which the mutation is undirected and the responsible gene is only determined when the mutant shows an interesting phenotype (Wagner et al., 2002). Forward genetics has been performed using chemical mutagenesis, but the method is inefficient and it is time consuming to localize the mutated gene responsible for a distinct phenotype. Therefore, this approach is not very often used and reverse genetics is the method of choise. More than 10 years ago it was shown that reverse genetics can be performed with EBV using cosmid vectors in eukaryotic cells (Tomkinson and Kieff, 1992). A major disadvantage of this method is that eight recombination events between the cosmids must occur in the cell to reconstitute recombinant virus, with the recombination frequency being much lower in eukaryotic cells compared to e.g. yeast or bacteria.
Another problem is unwanted recombination events within the virus genome during reconstitution (Spate et al., 1996). Finally, the establishment of revertants is very time consuming with the cosmid approach and therefore almost impossible/unfeasible.
Recently, genetic analysis of herpesviruses like EBV has been revolutionized by using bacterial artificial chromosomes (BACs). BACs are single copy F-factor-plasmid based vectors with a cloning capacity of more than 300 kb in size (Kim et al., 1992;
Shizuya et al., 1992). The strict control of the F-factor replicon maintains a single copy of the BAC per bacteria cell, reducing the risk of recombination via homologous DNA stretches present in multiple copies of the viral DNA insert. The advantage of this method is that a gene can be studied in the context of the whole genome. The first viral genome to be cloned into a BAC was the mouse cytomegalovirus genome (Messerle et al., 1997). The EBV strain B95.8 was used for cloning the whole EBV genome into a F-factor plasmid for genetic manipulation in E.coli (Delecluse et al., 1998). For selection in prokaryotic as well as eukaryotic cells the chloramphenicolacetyl transferase and hygromycin phospho-transferase genes were inserted. In addition, this EBV-BAC plasmid (p2089) (Fig.1.1), also called Maxi-EBV, additionally carries the green fluorescence protein (GFP) gene under the control of the CMV promoter, which makes identification of successfully transfected and infected cells possible. Infectious virus particles can be obtained from HEK293 cells by stable transfection with p2089 and induction of the lytic cycle. These p2089-derived virus particles infect and transform human primary B-lymphocytes and hence possess all the properties of wild-type EBV (Delecluse et al., 1998). The advantages of this
Introduction 10method is that any gene of interest can be mutated via homologous recombination in E.coli and the manipulated genome can be tested for successful mutagenesis before virus reconstitution by transfection of eukaryotic cells with the mutated Maxi-EBV.
This method is a fast one step procedure that also allows establishment of revertants in a reasonably short time period.
1.2 In vitro infection of primary human B-lymphocytes as a model system for EBV induced B-cell immortalisation 1.2.1 Formation of lymphoblastoid cell lines For the development of therapeutics a better understanding of the molecular mechanisms by which EBV infects and immortalises B-lymphocytes is important.
Knowledge of the proteins involved and their mechanism of action could be used for the development of specific inhibitors or vaccines against EBV.