Purification and characterization of the epstein‐barr virus nuclear antigen 2 using monoclonal antipeptide antibodies

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Abstract
The Epstein‐Barr virus (EBV) nuclear antigen 2 (EBNA‐2) is the only one of the EBNA proteins to have been implicated as an EBV‐encoded transforming protein. More detailed studies of this protein have been hampered by the lack of EBNA‐2‐specific monoclonal antibodies (MAbs) and of purified protein. To overcome these problems, we isolated 5 hybridomas producing MAbs reactive with an 18 residue synthetic peptide corresponding to the carboxyterminus of EBNA‐2. Four of the 5 MAbs were specifically reactive with EBNA‐2 in its denatured form on immunoblots. The 5th antibody (IISE) was reactive with the native form of EBNA‐2. By using a one‐step immunoaffinity purification method with II5E cross‐linked to protein‐A‐Sepharose, we purified EBNA‐2 to homogeneity, i. e., more than 1,200‐fold, from Burkitt lymphoma cell extracts. A major 32‐kDa associated protein and a less abundant 17‐kDa protein were co‐purified with EBNA‐2. Immunoprecipitation with 115E from 35S‐methionine‐labelled cell extracts showed that the 32‐kDa protein co‐precipitated with EBNA‐2 from EBV‐positive cells, but was not detectable in immunoprecipitates of EBV‐negative cells. When the immunoprecipitates or the purified proteins were immunoblotted with EBV‐immune sera, only EBNA‐2 was reactive, indicating that the associated proteins are of cellular origin. Immunoprecipitation of cells labelled with 32P‐orthophosphate showed that EBNA‐2, but not the associated proteins, is a phosphoprotein. The expression level of EBNA‐2 varied between different EBV‐carrying cell lines, as measured by a 2‐site ELISA based on antibody II5E. In indirect immunofluorescence, the II5E MAb gave an EBNA‐2‐specific characteristic granular staining pattern. These characteristics of EBNA‐2 resemble those of other viral transforming proteins.