RBP-J dependent and independent signalling of EBNA-2

EBNA-2 is a multifunctional viral oncogene involved in the
immortalisation of B-cells by EBV. EBNA-2 regulates transcription
of viral and cellular genes in the proliferative phase of the viral
life cycle, which in vitro results in the outgrowth of EBV positive
B-cells into lymphoblastoid cell lines (LCLs). EBNA-2
transcriptional signalling is mediated by cellular DNA-binding
proteins, such as RBP-J and PU.1, since EBNA-2 does not contain its
own DNA-binding domain. In order to better characterise EBNA-2
signalling we conducted a mutational analysis of the viral LMP-1
promoter that is strongly induced by EBNA-2 in the EBV-immortalised
B-cells. Our mutational analysis of the LMP-1 promoter confirmed
that the PU.1 binding site is important for transactivation of the
LMP-1 promoter by EBNA-2, whereas RBP-J binding to the LMP-1
promoter leads to repression and EBNA-2 binding to RBP-J is not
required for transactivation. These results imply that EBNA-2
transactivates the LMP-1 promoter preferentially by an RBP-J
independent mechanism. We further characterised EBNA-2 signalling
by dissection of promoter targeting domains in the EBNA-2 protein.
Two EBNA-2 mutants, the CR4del and WW mutant, preferentially
activated RBP-J dependent and independent signalling indicating
that EBNA-2 uses at least two separate signalling pathways. We
introduced the characterised EBNA-2 mutants into the EBV genome and
produced recombinant viruses carrying specific mutations in the
EBNA-2 genes. Primary B-cells were infected with increasing titres
of recombinant EBVs lacking the EBNA-2 ORF or carrying the WW or
CR4del mutant. Viruses lacking the EBNA-2 ORF or carrying the WW
mutant were not able to immortalise primary B-cells even at high
viral titres. The CR4 region of EBNA-2 strongly influenced B-cell
immortalisation efficiency and growth rate of the immortalised
B-cells. These results indicate that EBNA-2 and the RBP-J
signalling of EBNA-2 are absolutely essential for B-cell
immortalisation by EBV. In contrast, the CR4 EBNA-2 region
mediating RBP-J independent signalling is critical, but not
absolutely essential for the process of EBV immortalisation.