B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2.

Abstract

Infection of primary B‐lymphocytes by Epstein‐Barr virus (EBV) leads to growth transformation of these B‐cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B‐cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B‐cells by mutant virus resulted in estrogen‐dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non‐proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B‐cells. These data suggest that EBV is using a common pathway for B‐cell activation bypassing the requirement for antigen, T‐cell signals and growth factors.

Antagonistic effects ofc-myc and Epstein-Barr virus latent genes on the phenotype of human B cells

“…In EREB2-5 cells EBNA2 has a lower electrophoretic mobility and is present in higher quantity due to phosphorylation and stabilization of the protein in the presence of estrogen. 9 Other c-myc/Ig transfected EREB2-5 derivative cell lines proliferating in the absence of hormone showed similar growth pattern, proliferation kinetics, and phenotype as A1 cells, ruling out the possibility that A1 is a rare clonally selected variant (data not shown). 14 FIGURE 4 -Expression of cell surface markers in P493-6 cells in the various EBNA2 and MYC on and off conditions.…”

“…The cell lines EREB2-5 9 and A1 14 were established as described. The cell line P493-6 was established by stable transfection of EREB2-5 cells with a construct allowing tetracycline-regulatable expression of c-myc.…”

“…As a first step, we have established lymphoblastoid cells (LCL) conditionally immortalized by EBV expressing an estrogen regulated version of EBNA2. 8,9 EBNA2, together with EBNA-LP, is the first gene expressed in cells infected by EBV and acts as a transcriptional regulator of viral and cellular genes involved in B cell immortalization. 10,11 These conditionally immortalized cells proliferate in the presence of estrogen and stop proliferating when estrogen is withdrawn.…”

Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation

“…This P493-6 cell line is particularly useful because proliferation can also be driven by an estrogeninducible Epstein-Barr virus (EBV) EBNA-2 gene (Kempkes et al, 1995), so that c-Myc expression can potentially be turned on or off without altering proliferation of these cells. P493-6 cells grown in tetracycline do not express detectible Myc, while cells grown in the absence of tetracycline express high Myc levels (Pajic et al, 2000).…”

Loss of functional pRB is not a ubiquitous feature of B-cell malignancies

“…As quiescent (Go) cells enter into the cell division cycle in response to relevant extracellular signals, two changes to pRb protein become apparent: the overall level of expression increases and the protein becomes phosphorylated on multiple sites (Sinclair et al, 1994(Sinclair et al, , 1998Hollyoake et al, 1995;Kempkes et al, 1995;Szekely et al, 1995;Cannell et al 1996). Altered expression of pRb has been described in primary samples derived from high grade non-Hodgkin's lymphoma (NHL) (Martinez et al, 1993) and expression is lost in 58% of centroblastic and Burkitt's lymphomas (Weide et al, 1994).…”