Purification and characterization of the Epstein-Barr virus nuclear antigen 2 using monoclonal antipeptide antibodies.
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 (115E) was reactive with the native form of EBNA-2. By using a one-step immunoaffinity purification method with 115E 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 115E. In indirect immunofluorescence, the 115E MAb gave an EBNA-2-specific characteristic granular staining pattern. These characteristics of EBNA-2 resemble those of other viral transforming proteins.
Dillner
J
J
Department of Medical Cell Genetics, Karolinska Institute, Stockholm, Sweden.
Wendel-Hansen
V
V
Kjellström
G
G
Kallin
B
B
Rosén
A
A
D000818:Animals; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000302:isolation & purification*; D002467:Cell Nucleus / Q000276:immunology; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D019309:Epstein-Barr Virus Nuclear Antigens; D005455:Fluorescent Antibody Technique; D004854:Herpesvirus 4, Human / Q000276:immunology*; D007163:Immunosorbent Techniques; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D008715:Methionine