ABSTRACT
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.
Subject(s)
Antigens, Viral/analysis , DNA-Binding Proteins/analysis , Animals , Antigens, Viral/immunology , Blotting, Western , Cell Line , DNA-Binding Proteins/immunology , Epstein-Barr Virus Nuclear Antigens , HeLa Cells , Histones/isolation & purification , Histones/metabolism , Humans , Molecular Weight , Moths , Phosphoproteins/metabolism , Precipitin Tests , Protein Binding , Viral Proteins/metabolismABSTRACT
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.
Subject(s)
Antigens, Viral/metabolism , Herpesvirus 4, Human/immunology , Protein Kinases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Antigens, Viral/genetics , Casein Kinases , Cell Line , Epstein-Barr Virus Nuclear Antigens , Humans , Molecular Sequence Data , Peptide Fragments/isolation & purification , Peptides/chemical synthesis , Peptides/metabolism , Phosphorylation , Recombinant Fusion Proteins/metabolism , Substrate SpecificityABSTRACT
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.
