DNA-binding studies of the Epstein-Barr virus nuclear antigen 2 (EBNA-2): evidence for complex formation by latent membrane protein gene promoter-binding proteins in EBNA-2-positive cell lines.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c-fgr, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Trans-activation of the TP1 promoter and of the BamHI C promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates trans-activation. To determine whether EBNA-2 also trans-activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides -135 to -176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein-protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher M(r) in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay.

Immunological detection of proteins associated with the Epstein-Barr virus nuclear antigen 2A.

The Epstein-Barr virus nuclear antigen 2A (EBNA-2A) has been strongly implicated in the EBV-mediated B-cell transformation process. Since EBNA-2A might exert this function through interaction with proteins of the infected cell, we studied the association of EBNA-2A with cellular proteins. Immunoprecipitation of EBNA-2A from 32P-labeled cell extracts separated by sucrose gradient centrifugation revealed the presence of phosphoproteins complexed with the two forms of the EBNA-2A sedimenting at 13 S and 34 S. Prominent bands were observed at 250, 170, 120, 110, 105, and 95 kDa with minor species at 78, 52, 45, 31, 26, 22 and 18 kDa. By "West-Western" or "Far-Western" blotting using EBNA-2A protein from insect cells as a probe we detected binding to proteins migrating with apparent molecular masses of about 200, 130, 110, 105, 95, and 31 kDa with minor species detectable at 90, 68, 50-55, 40, and 17 kDa. The protein with an apparent molecular mass of 31 kDa was identified by competition experiments as histone H1. Some of the EBNA-2A-complexed phosphoproteins, notably the proteins of 110 and 95 kDa, comigrated with the proteins detectable by "West-Western" analysis. The binding of EBNA-2A to the 130-kDa protein was stable against up to 1.5 M NaCl and could not be competed with histone H1. In a similar experiment, the less transforming EBNA-2B which is encoded by the subtype 2 virus bound to most of the proteins detected with EBNA-2A but with strongly reduced efficiency to the protein of 130 kDa indicating that this protein might be a target for EBNA-2 during EBV-mediated transformation.

Phosphorylation of the Epstein-Barr virus nuclear antigen 2.

A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic peptide corresponding to amino acids 464-476 of EBNA-2 as a substrate led to the incorporation of 0.69 mol phosphate/mol peptide indicating that only one of three potential phosphorylation sites within the peptide was modified. The most likely amino acid residues for phosphorylation by CK-2 are Ser469 and Ser470.

Biochemical characterization of Epstein-Barr virus nuclear antigen 2A.

The Epstein-Barr virus nuclear antigen 2A (EBNA-2A) was immunoprecipitated from latently Epstein-Barr virus-infected lymphocytes with a polyclonal serum raised against the EBNA-2A C terminus. The nucleus contained three subfractions of EBNA-2A which could be distinguished by their resistance to salt extraction: (i) a nucleoplasmatic fraction that was solubilized at 50 mM NaCl, (ii) a chromatin-associated fraction extractable at 1.5 M NaCl, and (iii) a nuclear matrix-associated fraction solubilized only by boiling with buffer containing 2% sodium dodecyl sulfate. The three subfractions were phosphorylated; it was demonstrated that the nucleoplasmatic and the chromatin-associated fractions were phosphorylated at serine and threonine residues. The half-life of the EBNA-2A protein was determined by cycloheximide treatment and by pulse-chase experiments and was found to be at least 24 h. The turnover of the phosphate residues bound to the two salt-soluble subfractions was determined to be approximately 6 to 9 h, suggesting a possible role of the phosphorylation in the regulation of the biological activity of EBNA-2A. Dephosphorylation of EBNA-2A resulted in an increased mobility of the protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and indicated the presence of differentially phosphorylated subclasses of the protein. Analysis of EBNA-2A by sucrose gradient centrifugation revealed the existence of two subclasses of complexed molecules which exhibited sedimentation coefficients of approximately 13S and 34S.

Immunological characterization of the Epstein-Barr virus phosphoprotein PP58 and deoxyribonuclease expressed in the baculovirus expression system.

The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells. The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E. coli. DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities. Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58. The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E. coli. The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, B95-8, M-ABA and BL74 induced by TPA and n-butyrate. The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0). Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera. Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC. In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.