Immunological detection of proteins associated with the Epstein-Barr virus nuclear antigen 2A.
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.
Grässer
F A
FA
Abteilung Virologie, Universitätskliniken des Saarlandes, Homburg, Germany.
Sauder
C
C
Haiss
P
P
Hille
A
A
König
S
S
Göttel
S
S
Kremmer
E
E
Leinenbach
H P
HP
Zeppezauer
M
M
Mueller-Lantzsch
N
N
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis* / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000276:immunology; D019309:Epstein-Barr Virus Nuclear Antigens; D006367:HeLa Cells; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D009036:Moths; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014764:Viral Proteins / Q000378:metabolism
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D006657:Histones; D010750:Phosphoproteins; D014764:Viral Proteins