Epstein-Barr Virus Nuclear Protein EBNA3A Is Critical for Maintaining Lymphoblastoid Cell Line Growth

Abstract

To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.

RNAs induced by Epstein–Barr virus nuclear antigen 2 in lymphoblastoid cell lines

“…To investigate whether 4HT could have residual effects on endogenous hormone receptors, RNAs that were up-regulated by 4HT in E2HTF LCLs were compared with RNAs that were up-regulated by 4HT in EBNA3AHT LCLs (15)…”

“…IB4 is a lymphoblastoid cell line transformed by the B95-8 type I EBV. Five newly established LCLs were transformed by B95-8 virus (2) or WT recombinant EcoA cosmidrescued P3HR1 EBV (15). E2HTF LCLs were established by transforming cord blood B lymphocytes with mutant recombinant EcoA cosmid-rescued P3HR1 EBV.…”

“…P3HR1 EBV-infected cells were used to generate recombinant EBVs (15). IB4 is a lymphoblastoid cell line transformed by the B95-8 type I EBV.…”

“…Concordant with sustained LMP1 protein levels, gel-shift activity with an NF-B probe did not change over these 48 h (data not shown). Lack of change in EBNA1 and EBNA3A protein levels is due to protein stability, whereas lack of change in EBNALP and LMP1 protein levels may be due to relatively high mRNA levels in LCLs (1,15,16).…”

Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

“…Also, activated Notch expression can partially substitute for EBNA2 in maintaining LCL growth (10). Surprisingly, EBNA3A and EBNA3C are each uniquely important for initial and continued LCL growth, whereas EBNA3B is not required (11)(12)(13)(14)(15)(16), although EBNA3B also alters cell gene transcription (17,18). EBNA3C uniquely up-regulates LMP1 (19)(20)(21)(22)(23)(24), CD21(CR2), TCL1A, and ITGA4 expression and represses JAG1, NCALD, p16, BIM, and FLNA expression (14-16, 18, 19, 25, 26).…”