Generierung von Transposonmutanten des Epstein-Barr-Virus zur Charakterisierung von Genfunktionen für die lytische Virusvermehrung und die virale Latenz

The Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with malignancies like Burkitt’s lymphoma and nasopharyngeal carcinoma. So far, the initial steps of tumor development and the role of the virus in this process have not yet become clear. The key for elucidation of the oncopathogenic effect may lie in the identification of viral genes because the functions of most EBV-encoded genes and gene products are not yet known. To approach this problems, the EBV genome has to be mutated and the mutants have to be characterized. In this work, a method for efficient transposon-mutagenesis of the EBV is presented. EBV mutants have been generated and characterized morphologically and functionally. Mutants with insertion mutations in the open reading frames BGLF-5 (EBV DNase) and BDLF-2 were generated by homologous recombination between mutated EBV subclones and wildtype virus from the cell line B95-8 and subsequently selected in Raji cells. B-lymphocytes from EBV negative donors were infected with the EBV-BGLF-5- and EBV-BGLF-2- mutants, viral gene expression was investigated by PCR (BZLF-1, BRLF-1, BMRF-1, BHRF-1, EBNA-1, -2, -3A-C, LMP1, -2) and immuno fluorescence assay (EA-D, LMP-1). The cells were analyzed morphologically and with regard to a modulated expression of surface markers (CD11a, CD23, CD30, CD40, CD44, CD54, CD58). Wildtype EBV infected cells and uninfected cells were used as controls. Both mutants expressed a subset of the investigated genes for a short period. An increased surface marker expression could not be detected and the cells did not become immortalized. In wildtype virus infected B-cells in contrast, all genes were expressed during the observation period. The expression of surface markers was increased and the cells became immortalized. This led to the conclusion that the expression of BGLF-5 and BDLF?2 influences the lytic cycle, the establishment of latency and immortalization of B-lymphocytes. Thus, both mutants express viral products that cause an immune response, but do not have the potential to immortalize the cells or to establish latency. This makes them candidates for an EBV live vaccine.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2004

Author dc:creator

• Peters, Michele

Contributors dc:contributor

• Kreuzaler, Fritz

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Epstein-Barr Virus
• Transposonmutagenese

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123639%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:62041