Murine Model System for EBV-related Diseases

Epstein-Barr Virus (EBV) is involved in several human malignancies via its latent gene products, which interact with cellular proteins and mimic discrete functions of cellular signalling pathways. Enigmatically, more than 90% of the human population carries this human tumour virus but virus-associated tumours are relatively rare. Most studies on EBV have been carried out in vitro and ex vivo on EBV-transformed human B cells or on human biopsies. Established in vivo model systems do not reflect the main aspects of EBV-associated diseases in humans. This limited tool set is the result of EBV’s inability to infect cells of non-human origin, which lack the surface receptor for EBV. My PhD work aimed at engineering a transgenic mouse, which carries a conditionally inactivated EBV genome. This study took advantage of the well-established techniques of mouse genetics in order to stably integrate the entire EBV genome into the murine genome. This approach would not only overcome the inability of EBV to infect animal cells but it would also permit to study the complete virus in an immunocompetent and easy-to-handle living organism. I undertook two routes to establish a transgenic mouse with the complete EBV genome inserted. One route was based on the site-specific integration into the hprt locus of murine embryonic stem cells. The other route engaged pronucleus microinjection of the EBV DNA into fertilized murine oocytes. In addition, the EBV genome was genetically manipulated prior to its introduction into murine cells. On the basis of the E.coli cloned EBV strain B95.8, I constructed a novel EBV mutant with unique features. This EBV targeting construct (InvTarg) allows conditional expression of EBV’s latent genes via a Cre/loxP system. Such approach prevents potentially adverse effects of EBV’s latent genes on embryonic development but allows their expression in almost any chosen cellular compartment for which specific Cre-expressing mice are available. The InvTarg recombinant EBV genome is 185 kb in size, based on a bacterial replicon, and therefore belonging to Bacterial Artificial Chromosomes (BACs). Two genetically modified and inversely oriented loxP sites were introduced in E.coli cells at the predetermined sites of the InvTarg, and the bracketed segment was inverted by Cre recombinase, disrupting transcription of almost all viral latent genes. In transgenic animals this inversion can be reverted and the latent genes can be re-activated at will by cross-breeding with Cre-expressing mouse (re-inversion). The ability of Cre to invert the big fragment was verified in infection experiments with human primary B cells. As expected, the ‘inverted’ EBV construct, such as InvTarg, failed to transform primary B cells, when the viral latent genes were not expressed. Despite sustained efforts, both gene delivery techniques did not lead to a transgenic mouse with the entire EBV genome inserted, but resulted in the integration of only subgenomic segments of the InvTarg recombinant EBV DNA. A number of technical problems were identified during this work, indicating more specific direction for further research. On the basis of the experience gained here, the project of an EBV transgenic mouse can be carried on. In addition, the InvTarg maxi-EBV conditional vector might be employed in other experimental conditions, like different cell types or distinct stages of cell differentiation, for studies on latent EBV genes

Restricted Expression of Epstein-Barr Virus Latent Genes in Murine B Cells Derived from Embryonic Stem Cells

Background: Several human malignancies are associated with Epstein-Barr virus (EBV) and more than 95 % of the adult human population carries this virus lifelong. EBV efficiently infects human B cells and persists in this cellular compartment latently. EBV-infected B cells become activated and growth transformed, express a characteristic set of viral latent genes, and acquire the status of proliferating lymphoblastoid cell lines in vitro. Because EBV infects only primate cells, it has not been possible to establish a model of infection in immunocompetent rodents. Such a model would be most desirable in order to study EBV’s pathogenesis and latency in a suitable and amenable host. Methodology/Principal Findings: We stably introduced recombinant EBV genomes into mouse embryonic stem cells and induced their differentiation to B cells in vitro to develop the desired model. In vitro differentiated murine B cells maintained the EBV genomes but expression of viral genes was restricted to the latent membrane proteins (LMPs). In contrast to human B cells, EBV’s nuclear antigens (EBNAs) were not expressed detectably and growth transformed murine B cells did not arise in vitro. Aberrant splicing and premature termination of EBNA mRNAs most likely prevented the expression of EBNA genes required for B-cell transformation. Conclusions/Significance: Our findings indicate that fundamental differences in gene regulation between mouse and ma