Epstein-Barr virus EBNA-3C is targeted to and regulates expression from the bidirectional LMP-1/2B promoter

Abstract

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is essential for EBV-mediated immortalization of human B lymphocytes and regulates both the cell cycle and transcription. Transient reporter gene assays have implicated a pivotal role for EBNA-3C in the regulation of transcription of the majority of latency-associated genes expressed during the EBV growth program, including the viral oncoprotein LMP-1. To examine the regulation of latency gene expression by EBNA-3C, we generated an EBV-positive cell line that inducibly expresses EBNA-3C. This cell line allowed us to examine expression from the endogenous latency gene promoters in the context of an actual latent infection and the presence of other EBNA proteins, in particular EBNA-2, which is presumed to coregulate transcription with EBNA-3C. EBNA-3C induced the expression of both LMP-1 and LMP-2B mRNAs from the bidirectional LMP-1/LMP-2B promoter. In contrast, no effect was seen on expression from the common EBNA promoter Cp, which is responsive to EBNA-3C in reporter assays. Activation of LMP expression was not the consequence of increases in EBNA-2, PU.1 or Spi-B transcription factors, all of which are believed to be critical for activation of LMP-1. Chromatin immunoprecipitation assays furthermore indicated that EBNA-3C is present at the bidirectional LMP-1/LMP-2B promoter. These results indicate that EBNA-3C directly activates the expression of LMP-1 and LMP-2B but is unlikely to significantly regulate EBNA expression via Cp under normal growth conditions.

FIG. 1.

EBNA-3C activates expression of the LMP-1 protein. Expression of EBNA-3C was induced by growing cells in the absence of doxycycline for 5 days. (A) Expression of EBNA-3C, LMP-1, and actin was determined by immunoblot analysis. (B) Cells that were not induced (left) or induced to express EBNA-3C (right) were examined by light microscopy.

FIG. 2.

Time course of EBNA-3C and LMP-1 induction. Raji Tet-off-3C cells (upper panel) were cultured in the presence or absence of doxycycline and harvested at different time points. SDS-polyacrylamide gel electrophoresis was performed in triplicate. Each immunoblot was stripped and reprobed for actin. Parallel experiments were performed with the Raji Tet-off parental cell line (lower panel). Mock induction refers to the absence of doxycycline (+), which would induce EBNA-3C in Raji Tet-off-3C cells; cells incubated in the presence of doxycycline, which would not induce EBNA-3C, are represented by “−.”

FIG. 3.

EBNA-3C does not affect the level of EBNA-3A. Raji Tet-off-3C cells were cultured for 48 h in the presence or absence of doxycycline. A sample of IB4 cells (an LCL) was included as a control. Immunoblots were performed for the indicated proteins and reprobed for actin.

FIG. 4.

EBNA-3C increases expression of LMP-1 mRNA. LMP-1 mRNA was detected by Northern blot analysis of total RNA isolated from Raji Tet-off cells expressing (+) or not expressing (−) EBNA-3C. The blot was stripped and reprobed for GAPDH as a loading control.

FIG. 5.

EBNA-3C activates expression of LMP-2B mRNA. (A) Schematic of LMP-1/LMP-2B bidirectional promoter. Binding sites for the transcription factors Jκ and PU.1 are shown. Boxes below the map depict the mRNAs for LMP-1 and LMP-2B (not to scale), showing coding (black boxes) and noncoding (white boxes) exons. (B) Raji Tet-off EBNA-3C cells were induced to express EBNA-3C, and cells were harvested 24 or 48 h later. Total RNA was isolated, and primers specific for LMP-2B, EBNA-3C, and GAPDH were used to determine the level of each mRNA by RT-PCR, using [α-³²P]dCTP in the reaction mix for quantification.

FIG. 6.

EBNA-3C does not affect the level of PU.1 or Spi-B. Extracts from Raji Tet-off cells expressing (+) or not expressing (−) EBNA-3C and from the parental cell line Raji Tet-off were analyzed for levels of the transcription factors PU.1 and Spi-B, which are key activators of the LMP-1 promoter.

FIG. 7.

EBNA-3C interacts with the bidirectional LMP-1/LMP-2B promoter. Raji Tet-off-3C cells were induced to express EBNA-3C by removal of doxycycline from the growth medium (+) for 48 h or were kept under doxycycline repression (−). A ChIP assay was performed to examine the association of EBNA-3C with the LMP-1/LMP-2B promoter (upper panel). The chromatin was immunoprecipitated using an antibody specific for EBNA-3C. As a positive control, the chromatin was immunoprecipitated with an antibody to TFIIB, a known element of the basal transcriptional machinery which associates with the GAPDH promoter (lower panel) and also with the LMP-1 promoter. As a negative control, the chromatin was immunoprecipitated with an irrelevant IgG. The GAPDH and LMP-1 primers correspond to PCR primers to amplify the promoter sequences of these genes. Negative control primers were designed to span sequences outside the GAPDH promoter area with which TFIIB is associated.