Regulation of CD95 in EBV infected B-Cells.

Expression of CD95 is regulated by various agents such as TNFa, IFNg, or Daunorubicine, able to induce the transcriptional factors NF-kB, STAT1 and p53 respectively. The CD95 promoter is known to harbour binding sites for these factors. These factors are modulated both in expression and activity during EBV infection of B-cells. EBV is known to induce CD95 expression. The objective of this work was to understand the contribution of p53, STAT1 and NF-kB in the regulation of CD95 expression by EBV.

To control the activity of NF-kB, STAT1, p53 and LMP1, we have developed and used various episomal inducible vectors. One of the characteristics of these vectors is that the gene of interest is expressed from a bidirectional doxycycline regulatable promoter that allowing simultaneous expression of truncated NGF receptor, used as a surrogate marker of inducibility. We have subcloned the following cDNAs into these vectors: dominant negative IkBa, active (STAT1a) and dominant inactive natural isoform of STAT1 (STAT1b), p53, a dominant negative mutant of LMP1 (LMP1CT) and LMP1 wild type. After stable transfection of an LCL, induction of the cDNA of interest was performed with doxycycline for 24H. Positive cells for NGFR were purified using magnetic beads and were additionally treated with TNFa, IFNg or Fludarabine for 24H. To control the latency III program, we have used the EREB2-5 cell line harboring an estrogen-regulatable EBNA2 gene. The function of CD95 was studied by quantifying apoptosis induction after CD95 cross linking with a specific antibody.

We found that induction of latency III program in EREB 2-5 cells was associated with an increase of both p53 and CD95 expression.

Inhibition of LMP1 by LMP1CT decreased the expression of CD95. CD95 expression was hardly regulated by p53. Inhibition of both NF-kB and STAT1 decreased CD95 expression. Only IFNg was able to increase expression of CD95. Finally, we show that induction of CD95 expression by EBV render the infected B-cell sensitive to the induction of CD95-mediated apoptosis. Our results suggest that the latencyc III program of EBV sensitize the infected B-cells to CD95-mediated apoptosis via STAT1 and NF-kB activated by LMP1. In the view of the virus/host equilibrium, this process may render EBV-infected B-cells in latency III susceptible to elimination by the immune system.