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.