Multiple functions within the Epstein-Barr virus EBNA-3A protein

Abstract

Two regions of the EBNA-3A protein of Epstein-Barr virus were shown to be capable of binding to the cell protein RBP-Jk (also known as CBF-1), a component of the Notch signaling pathway. Consistent with this binding, EBNA-3A inhibited reporter gene expression from plasmids containing RBP-Jk DNA binding sites within their promoters, including the Cp promoter. When EBNA-3A was linked to a GAL4 DNA binding domain, it repressed the activity of a promoter containing GAL4 binding sites at all plasmid concentrations tested. However, a deletion mutant of EBNA-3A lacking amino acids 100 to 364 showed a biphasic response in the GAL4 assay: it inhibited transcription at low DNA concentrations but activated it at high DNA concentrations. There appears to be a gene activation function within EBNA-3A that is masked in the full-length protein in this assay. Current models for EBNA-3 function have stressed transcription repression through binding to RBP-Jk, but we consider an alternative scheme in which the role of the binding of EBNA-3A, -3B, and -3C to RBP-Jk is to buffer the levels of active EBNA-3 protein. We have also found that the behavior of EBNA-3A in a cell fractionation procedure that distinguishes insoluble matrix from soluble cell fractions is modified by EBNA-LP, indicating a further novel level of interplay between the EBNA proteins.

FIG. 1

GST pull-down assay identifying two regions within EBNA-3A capable of binding to RBP-Jk. (A) In vitro-translated proteins analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. This film was exposed for one-seventh the time used in panels B and C. (B) In vitro-translated EBNA-3A proteins that bound to GST-RBP-Jk in a binding assay performed at 4°C. (C) Negative control binding of in vitro-translated EBNA-3A proteins to GST at 4°C. Positions of protein size markers are shown in kilodaltons. (D) EBNA-3A and deletion mutants. Numbers are the amino acids of the 944-amino-acid EBNA-3A protein present, except for Δ100-364, in which residues 100 to 364 are absent. The percentage of input protein that bound to GST-RBP-Jk in panel B was quantified by densitometry and is shown on the right. (E) Time course of GST-RBP-Jk binding with EBNA-3A mutants at 30°C. The bound EBNA-3A protein was quantified by PhosphorImager analysis and expressed as a percentage of the input protein.

FIG. 2

Repression of promoters containing RBP-Jk binding sites by EBNA-3A. The results in panels A to C are expressed as relative CAT activity, the CAT activity with the empty vector pSNOC being set at 100. (A) EBNA-3A inhibits Cp in DG75 cells. pSNOC-E3A (0.5 to 5 μg) was titrated on p1.4Cp-CAT (black bars) or p1.4Cp-CAT E2RE mut (grey bars). (B) EBNA-3A inhibits Cp in 293 cells. pSNOC-E3A (0.1 to 2.5 μg) was titrated as in panel A. (C) EBNA-3A inhibits a promoter containing four RBP-Jk binding sites. pSNOC-E3A (0.5 to 5 μg) was titrated in DG75 cells on 4Jk-TK-CAT (black bars) or 4Jk-TK-CAT E2RE mut (grey bars). (D) Western immunoblot analysis of EBNA-3A expression. Samples from panel B were analyzed with antibody T2.78. EBNA-3A concentrations are given in micrograms.

FIG. 3

Comparison of transcription regulation by EBNA-3A and EBNA-3A(Δ100-364) in the GAL4 system. The results in panels A to D are expressed as relative CAT activity, the CAT activity with vector pGAL4(1-147) being set at 100. (A) GAL4-E3A inhibits transcription from a GAL4-dependent promoter in transfected DG75 cells. GAL4-E3A (0.5 to 5 μg) was titrated on pUASCAT (5 μg; black bars) or pBLCAT2 (5 μg; grey bars). (B) Effect of GAL4-E3A(Δ100-364) on transcription in transfected DG75 cells. Symbols are as in panel A. (C) GAL4-E3A and GAL4-E3A(Δ100-364) both inhibit transcription from pUASCAT in transfected HeLa cells (5 μg of pUASCAT and 5 μg of GAL effector DNA were transfected). (D) GAL4-E3A and GAL4-E3A(Δ100-364) both inhibit transcription from pUASCAT in transfected 293 cells (5 μg of pUASCAT and 5 μg of GAL effector DNA were transfected). (E) Western blot analysis of GAL4-E3A and GAL4-E3A(Δ100-364) expression in transfected DG75 cells. Samples from panels A and B were assayed with GAL4 monoclonal antibody sc-510. The arrows indicate the GAL4-E3A and GAL4-E3A(Δ100-364) proteins. (F) Western blot of extracts of transfected 293 cells with sc-510 anti-GAL4 (left panel) or T2.78 anti-EBNA-3A (right panel) antibodies. Lanes: 1, GAL4(1-147); 2, GAL4-E3A; 3, GAL4-E3A(Δ100-364). Only the high-molecular-weight EBNA-3A region of the gel is shown, so the small GAL4(1-147) protein is not seen in lane 1 of the left panel.

FIG. 4

EBNA-3A, a nuclear protein, is relocated to the nuclear matrix fraction by transfection of EBNA-LP. Western immunoblotting for the indicated proteins was done with extracts of DG75 cells transfected with combinations of EBNA expression plasmids. RIPA buffer extracts of transfected cells were centrifuged, yielding a soluble fraction (S) and a pellet (P). Where indicated (sonic), the RIPA buffer extracts were sonicated before centrifugation. Standard RIPA buffer extractions were done for 30 min, but in the panel marked with an asterisk, the extractions were done for 60 min. In each case (right-hand panels), expression of the transfected EBNA protein was demonstrated by comparison of cells transfected with the EBNA plasmid (+) and cells transfected with the corresponding empty vector (−).