Epstein-Barr virus nuclear antigen 3C putative repression domain mediates coactivation of the LMP1 promoter with EBNA-2 - PubMed (original) (raw)

Epstein-Barr virus nuclear antigen 3C putative repression domain mediates coactivation of the LMP1 promoter with EBNA-2

Jeffrey Lin et al. J Virol. 2002 Jan.

Abstract

The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) regulates virus and cell genes and is essential for EBV-mediated transformation of primary B lymphocytes. EBNA-3C associates with the cellular DNA sequence-specific transcription factors RBP-Jkappa and PU.1 and coactivates the EBV LMP1 promoter with EBNA-2 in BL2 and Raji cells under conditions of restrictive growth. We now find that EBNA-3C is similar to EBNA-LP in coactivating the LMP1 promoter with EBNA-2 in non-EBV-infected Burkitt lymphoma cells under conditions of maximal cell growth, whereas the EBV Cp promoter is repressed under the same conditions. EBNA-3A and EBNA-3B coactivation are at most 40% that of EBNA-3C. The RBP-Jkappa binding sites of EBNA-2 and the LMP1 promoter are not required for EBNA-3C coactivation, whereas the PU.1 site in the LMP1 promoter is required for EBNA-2-mediated activation and EBNA-3C coactivation. EBNA-3C amino acids (aa) 365 to 545, including most of the previously identified repression domain (M. Bain, R. J. Watson, P. J. Farrell, and M. J. Allday, J. Virol. 70:2481-2489, 1996), are necessary and sufficient for coactivation with wild-type EBNA-2. EBNA-3C can also coactivate with the EBNA-2 acidic activating domain; this activation does not require aa 343 to 545. These data indicate that there are at least two mechanisms by which EBNA-3C coactivates the LMP1 promoter with EBNA-2. Of the proteins that interact with EBNA-3C in a yeast two-hybrid screen, only the ubiquitin-like proteins SUMO-1 and SUMO-3/hSMT3B map to aa 365 to 545, implicating these molecules in EBNA-3C coactivation. In addition, SUMO-1 associates at a high level with EBNA-3C in lymphoblasts. Promoter coactivation by EBNA-3C is likely to be important in ensuring adequate levels of LMP1, while inhibition of the EBNA-Cp promoter under the same conditions prevents uncontrolled up-regulation of EBNA expression from a positive-feedback loop.

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Figures

FIG. 1.

FIG. 1.

Schematic diagram of wild-type EBNA-3C and deletion mutants used in these experiments. (A) aa 90 to 320 have the highest level of identity (22 to 27%) among the EBNA-3 proteins. The RBP-Jκ-binding site (aa 181 to 257), a PU.1-binding region (aa 181 to 365) (64), and a putative leucine zipper (aa 239 to 285) are indicated. The repression (aa 346 to 543) and activation (aa 724 to 826) domains were defined based on their effects on a Gal4-responsive promoter when fused to the Gal4 DBD (3, 31). Repeats containing prolines (PP) and glutamine-proline residues (QP) are noted, as are two potential nuclear localization signal sequences (NLS). (B) EBNA-3C deletion constructs utilized in this study. (C) Immunoblots of lysates from 12 million BJAB cells that had been transfected 40 to 48 h previously with 1 μg of pSG5 expression vector for the indicated EBNA-3C deletion construct. The left panel used the EBNA-3C-specific A10 monoclonal antibody, while the right panel used M2 and M5 monoclonal antibodies against the Flag epitope tag. The top arrow indicates the position of Flag-EBNA-3C aa 363 to 992, which migrates just below a background band present in both lanes, and the bottom arrow indicates the position of Flag-EBNA-3C aa 365 to 545. WB, Western blot; wt, wild type.

FIG. 2.

FIG. 2.

EBNA-3C potentiates EBNA-2 activation of the LMP1 promoter in BJAB, a non-EBV-infected BL cell line. (A) BJAB cells were transfected with reporter plasmid p(−512/+72)LMP1p-Luc, which has two tandem copies of the −512/+72 LMP1 promoter upstream of the luciferase gene, in addition to the indicated amounts of the pSG5-EBNA-2 (E2) and pSG5-EBNA-3C (E3C) expression vectors. Fold increase in luciferase activity is shown on the left. The panels below the graph show corresponding immunoblots using a monoclonal antibody against EBNA-2 (PE2). The results are representative of at least two independent experiments. WB, Western blot. (B) Response of p(−512/+72)LMP1p-Luc activity to 1 μg of pSG5-EBNA-2 and various increasing amounts (0.1 to 20 μg) of pSG5-EBNA-3C. The values shown are representative ratios of observed luciferase activity relative to that of EBNA-2 alone from at least two experiments. (C) Representative reporter assay using BJAB cells transfected with a reporter construct (pLuc-Cp) containing eight copies of the RBP-Jκ-binding site from the Cp promoter, 1 μg of pSG5-EBNA-2, and various amounts of pSG5-EBNA-3C. The relative luciferase values, indicated on the left in all experiments, were normalized to the β-galactosidase activity of a cotransfected plasmid, pGK-βgal.

FIG. 3.

FIG. 3.

EBNA-3C coactivation of the LMP1 promoter element is independent of RBP-Jκ. (A) Representative assay (of two repetitions) using BJAB cells transfected with the reporter plasmid p(−236/−145)LMP1p-Luc containing the −236/−145 LMP1 promoter element upstream of a minimal SV40 early promoter and the luciferase ORF, along with the indicated amounts of pSG5-EBNA-2 (E2) and pSG5-EBNA-3C (E3C). Fold increase in luciferase activity is indicated. (B) BJAB cells were transfected with CAT reporter plasmids containing wild-type (wt) or mutant LMP1 promoter sequences and the indicated amounts of pSG5-EBNA-2 and pSG5-EBNA-3C. Results are representative of two independent repetitions. Percent conversion was determined using ImageQuant software and a PhosphorImager. Fold activity is relative to that of the vector-only control, which was assigned a value of 1. (C) BJAB cells were transfected with p(−512/+72)LMP1p-Luc and the indicated amounts of pSG5-EBNA-2-SS (E2-SS) and pSG5-EBNA-3C. Results are representative of independent duplicate experiments. Fold activation of luciferase is indicated. (D) BJAB cells were transfected with p(−512/+72)LMP1p-Luc and the indicated amounts of pSG5-EBNA-2 and pSG5-flagEBNA-3C, pSG5-flagEBNA-3A, or pSG5-flagEBNA-3B. The results are representative of two independent experiments. Relative luciferase activity is indicated. The panel below the graph shows a corresponding immunoblot with monoclonal antibodies directed against the Flag epitope tag. WB, Western blot.

FIG. 4.

FIG. 4.

Amino acid residues 365 to 545 of EBNA-3C are necessary and sufficient for coactivating activity with EBNA-2. Reporter assay in BJAB cells using p(−512/+72)LMP1p-Luc, 1 μg of pSG5-EBNA-2 (E2), and 2 μg of either pSG5-EBNA-3C (E3C) or the indicated EBNA-3C deletion construct. Relative luciferase activity is indicated. The results are averages of duplicate samples and are representative of at least three independent repetitions. Error bars indicate standard deviations.

FIG. 5.

FIG. 5.

EBNA-3C can enhance transcriptional activity of the EBNA-2AD (aa 426 to 462) but not the VP16AD. (A) BJAB cells were transfected with reporter plasmid pFR-Luc, which contains five Gal4-binding sites, and the indicated amounts of pGal4-EBNA-2AD (E2-AD) and pSG5-EBNA-3C (E3C). The results from a representative experiment are depicted. (B) BJAB cells were transfected with pFR-Luc and the indicated amounts of pGal4-VP16 (VP16-AD) and pSG5-EBNA-3C. The results from a representative experiment are depicted. Luciferase activities are shown relative to those of reporter and empty expression vector control.

FIG. 6.

FIG. 6.

EBNA-3C coactivation of EBNA-2 and the EBNA-2AD are distinct activities. (A) Typical results from a reporter assay in BJAB cells using the pFR-Luc reporter construct and the indicated amounts of pGal4-EBNA-2AD (E2-AD) and pSG5-flagEBNA-3C (flag-EBNA3C), pSG5-flagEBNA-3A (flag-EBNA3A), or pSG5-flagEBNA-3B (flag-EBNA3B). (B) Reporter assay in BJAB cells using pFR-Luc, 1 μg of pGal4-EBNA-2AD, and 5 μg of either pSG5-EBNA-3C (E3C) or the indicated EBNA-3C deletion construct. The results are averages of duplicate samples and are representative of at least three independent repetitions. Error bars indicate standard deviations. Luciferase activities are shown relative to those of reporter and empty expression vector control.

FIG. 7.

FIG. 7.

EBNA-3C, which binds ubiquitin-like proteins SUMO-1 and SUMO-3/hSMT3b in yeast, can interact with these proteins in vitro and in cells. (A) GST-binding assay with the indicated GST fusion protein and in vitro-transcribed-translated [35S]-labeled EBNA-3C (IVT E3C). The first lane contains 10% of the amount of input EBNA-3C applied to each binding reaction, and subsequent lanes show the results of binding to the indicated GST fusion proteins. Coomassie staining confirmed that the GST control protein was used at a >5-fold excess relative to the GST fusion proteins (data not shown). (B) BJAB cells were transiently transfected with 10 μg of pSG5-EBNA-3C (EBNA3C) and/or pSG5-flagSUMO-1 (flag-SUMO-1) as indicated. Immunoprecipitations (IP) from these cell lysates were performed using rabbit antiserum reactive against EBNA-3C (R1.9), followed by immunoblotting with EBNA-3C-reactive monoclonal antibody (A10) and anti-Flag monoclonal antibodies (M2/M5). WB, Western blot.

FIG. 8.

FIG. 8.

Proposed roles of EBNA-3C in regulating EBNA-2 transactivation activity in EBV-infected B lymphocytes. The Wp EBNA promoter directs initial transcription of EBNA-2 and EBNA-LP mRNAs, which are polyadenylated at a site downstream of the EBNA-2 ORF. EBNA-2 activates the Cp promoter and probably also up-regulates the Wp promoter, resulting in higher-level transcription through EBNA-LP and EBNA-2 and transcription of EBNA-3A, EBNA-3B, EBNA-3C, and EBNA-1 mRNAs. EBNA-2, EBNA-LP, and EBNA-3C collaborate to coactivate the LMP1 promoter and to sustain LMP1 expression at the appropriate level under all conditions of cell growth. LMP1 has a key role in LCL transformation, growth, and survival. EBNA-2, EBNA-LP, and EBNA-3C could cause runaway positive feedback up-regulation of the EBNA Cp and Wp promoters, but EBNA-3C, EBNA-3A, and EBNA-3B all contribute to repressing EBNA-2 and EBNA-LP coactivation of these promoters. Similar effects of EBNA-2, EBNA-LP, EBNA-3C, EBNA-3A, and EBNA-3B are presumed to govern transcription at other viral and cell promoters in latent EBV infection.

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