BIOCHEMICAL-CHARACTERIZATION OF EPSTEIN-BARR-VIRUS NUCLEAR ANTIGEN 3AAND 3C PROTEINS

Among the viral proteins expressed in Epstein-Barr virus (EBV)-infecte d B cells are a family of nuclear proteins known the Epstein-Barr viru s nuclear antigen 3 (EBNA-3) proteins. Two of these, EBNA-3A and EBNA- 3C, have an essential but uncharacterized role in the transformation o f primary a cells by EBV. EBNA-3C increases expression of two genes li kely to be important for a cell growth transformation by EBV, the a ce ll activation antigen CD21 and the EBV latent membrane protein-1. Sinc e EBNA-3 proteins exhibit DNA-binding capability in crude protein extr acts from EBV-transformed cell lines and EBNA-3C contains sequences ho mologous to a basic leucine zipper motif found in one class of mammali an transcription factors, it is likely that EBNA-3C functions as a tra nscriptional transactivator. in this paper, we have overexpressed EBNA -3A and -3C in the baculovirus-expression system. To determine whether the ability to bind to DMA is an intrinsic property of the EBNA-3 pro teins, we have examined the ability of the recombinant protein to bind to DNA-cellulose. Unlike EBNA-3 proteins in lysates from EBV-transfor med cells, neither recombinant protein exhibits significant DNA-bindin g 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 phosphorylat ion status of the EBNA-3 proteins both in EBV-transformed cells and in infected insect cells. EBNA-3A and EBNA-3C were phosphorylated in bot h cell types. Therefore, if indeed these proteins function as transcri ptional transactivators, they may bind to DNA via an indirect mechanis m. The recombinant proteins will be invaluable in the further clarific ation of the role of EBNA-3A and EBNA-3C in EBV-induced immortalizatio n of B cells. (C) 1994 Academic Press, Inc.