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. 2005 May;79(9):5880-5.
doi: 10.1128/JVI.79.9.5880-5885.2005.

Hyperphosphorylation of EBNA2 by Epstein-Barr virus protein kinase suppresses transactivation of the LMP1 promoter

Wei Yue  1 , Edward Gershburg, Joseph S Pagano
Affiliations

Hyperphosphorylation of EBNA2 by Epstein-Barr virus protein kinase suppresses transactivation of the LMP1 promoter

Wei Yue et al. J Virol. 2005 May.

Abstract

The Epstein-Barr virus (EBV) BGLF4 gene encodes a serine/threonine protein kinase (PK) that is expressed in the cytolytic cycle. EBV nuclear antigen 2 (EBNA2) is a key latency gene essential for immortalization of B lymphocytes and transactivation of viral and cellular promoters. Here we report that EBV PK phosphorylates EBNA2 at Ser-243 and that these two proteins physically associate. PK suppresses EBNA2's ability to transactivate the LMP1 promoter, and Ser-243 of EBNA2 is involved in this suppression. Moreover, EBNA2 is hyperphosphorylated during EBV reactivation in latently infected B cells, which is associated with decreased LMP1 protein levels. This is the first report about the effect of EBV PK on the function of one of its target proteins and regulation of EBNA2 phosphorylation during the EBV lytic cycle.

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Figures

FIG. 1.
FIG. 1.
EBV PK phosphorylates EBNA2 at Ser-243. (A) Hyperphosphorylation of EBNA2 when coexpressed with EBV PK in vivo. HeLa cells were transfected with pSG5-EBNA2 alone or cotransfected with pcDNA-BGLF4-FLAG and collected 48 h after transfection. Left panel, immunoblotting for EBNA2; right panel, EBNA2 immunoprecipitated with EBNA2 antibody from the cell lysates was subjected to immunoblotting directly (−) or after treatment with 2,000 units of λ-PPase (+). (B) In vitro kinase assay. Phosphorylation of purified GST-EBNA2 185-315aa (GST E2) or GST-EBNA2 185-315aa S243A (GST E2 S243A) by EBV PK (PK) or kinase-dead PK (mt PK) was carried out. The whole reaction mixtures were separated by SDS-polyacrylamide gel electrophoresis (4 to 20% gradient gels); after CBB staining, gels were exposed on a PhosphorImager. (C) Involvement of Ser-243 in phosphorylation of EBNA2 by PK in vivo. pSG5-EBNA2 wild type or the S243A mutant of EBNA2 was coexpressed with EBV PK or empty vector in HeLa cells. Whole-cell lysates were subjected to immunoprecipitation with EBNA2 antibody, washed extensively, blotted, and probed with mouse monoclonal phosphoserine antibody or EBNA2 antibody.
FIG. 2.
FIG. 2.
Coimmunoprecipitation of EBNA2 and EBV PK. HeLa cells were transfected with pSG5-EBNA2 (EBNA2) (lanes 7, 8, 9) or pcDNA-BGLF4-FLAG (PK) alone (lanes 4, 5, 6) or cotransfected with both plasmids (EBNA2+PK) (lanes 1, 2, 3). Transfection with pcDNA3 served as a negative control (lanes 10, 11, 12). Cells were collected 48 h posttransfection, and aliquots of cell lysates from each transfection were subjected to immunoprecipitation with antibodies against EBNA2 or FLAG; normal mouse immunoglobulin G was used as a negative control. Immunocomplexes were resolved in 10% SDS-polyacrylamide gel electrophoresis, and immunoblots were carried out for EBNA2 and FLAG from the same gel. Whole-cell lysates before immunoprecipitation served as an input control (lanes 13, 14, 15, 16), and β-actin was used as a loading control.
FIG. 3.
FIG. 3.
Phosphorylation of EBNA2 by EBV PK suppresses transactivation of the LMP1 promoter. (A) LMP-1 promoter construct pGL2 (−512/+72)-luciferase was cotransfected with pSG5 EBNA2 (EBNA2), pHD/BGLF4 (PK), or pHD/K128Q (PK K128Q) in different combinations; pRL-TK plasmid was cotransfected as an internal standard. Levels of transactivation of the LMP-1 promoter are normalized to the vector control. Each data point represents the average of three independent experiments, each done in triplicate. Error bars represent the means ± standard errors. Immunoblotting shows the EBNA2 expression level in each transfection; β-actin was used as a loading control. (B) The EBNA2 S243A mutant was tested similarly as detailed in panel A.
FIG. 4.
FIG. 4.
Hyperphosphorylation of EBNA2 and decreased LMP1 protein level during EBV reactivation. The tetracycline-inducible cell line B95-8 BZLF1 was induced with doxycycline at a final concentration of 1 μg/ml for 72 h, and immunoblots of EBNA2, LMP1, and EA-D were carried out in induced (Id) and noninduced (N-Id) cells. β-Actin served as a loading control.
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