Biochemical characterization of Epstein-Barr virus nuclear antigen 3A and 3C proteins.
Among the viral proteins expressed in Epstein-Barr virus (EBV)-infected B cells are a family of nuclear proteins known as the Epstein-Barr virus nuclear antigen 3 (EBNA-3) proteins. Two of these, EBNA-3A and EBNA-3C, have an essential but uncharacterized role in the transformation of primary B cells by EBV. EBNA-3C increases expression of two genes likely to be important for B cell growth transformation by EBV, the B cell activation antigen CD21 and the EBV latent membrane protein-1. Since EBNA-3 proteins exhibit DNA-binding capability in crude protein extracts from EBV-transformed cell lines and EBNA-3C contains sequences homologous to a basic leucine zipper motif found in one class of mammalian transcription factors, it is likely that EBNA-3C functions as a transcriptional transactivator. In this paper, we have overexpressed EBNA-3A and -3C in the baculovirus-expression system. To determine whether the ability to bind to DNA is an intrinsic property of the EBNA-3 proteins, we have examined the ability of the recombinant protein to bind to DNA-cellulose. Unlike EBNA-3 proteins in lysates from EBV-transformed cells, neither recombinant protein exhibits significant DNA-binding capability as evidenced by the inability to bind to double-stranded DNA-cellulose. Since this difference in DNA binding could be a result of post-translational modifications, we have examined the phosphorylation status of the EBNA-3 proteins both in EBV-transformed cells and in infected insect cells. EBNA-3A and EBNA-3C were phosphorylated in both cell types. Therefore, if indeed these proteins function as transcriptional transactivators, they may bind to DNA via an indirect mechanism. The recombinant proteins will be invaluable in the further clarification of the role of EBNA-3A and EBNA-3C in EBV-induced immortalization of B cells.
Department of Virology and Molecular Biology, St Jude Children's Research Hospital, Memphis, Tennessee 38105.
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D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D016367:Baculoviridae; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D009036:Moths; D010766:Phosphorylation; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D011993:Recombinant Fusion Proteins