Deiminated Epstein-Barr virus nuclear antigen 1 is a target of anti-citrullinated protein antibodies in rheumatoid arthritis

Objective. To test the hypothesis that deimination of viral sequences containing Arg-Gly repeats could generate epitopes recognized by anti-citrullinated protein antibodies (ACPAs) that are present in rheumatoid arthritis (RA) sera. Methods. Multiple antigen peptides derived from Epstein-Barr virus (EBV)-encoded Epstein-Barr nuclear antigen 1 (EBNA-1), were synthesized, substituting the arginines with citrulline, and were used to screen RA sera. Anti-cyclic citrullinated peptide antibodies were purified by affinity chromatography and tested on a panel of in vitro deiminated proteins. Their ability to bind in vivo deiminated proteins was evaluated by immunoprecipitation, using EBV-infected cell lines. Results. Antibodies specific for a peptide corresponding to the EBNA-]([35-58) sequence containing citrulline in place of arginine (viral citrullinated peptide [VCP]) were detected in 50% of RA sera and in <5% of normal and disease control sera. In addition, affinity-purified anti-VCP antibodies from RA sera reacted with filaggrin-derived citrullinated peptides, with deiminated fibrinogen, and with deiminated recombinant EBNA-1. Moreover, anti-VCP antibodies immunoprecipitated, from the lysate of calcium ionophore-stimulated lymphoblastoid cell lines, an 80-kd band that was reactive with a monoclonal anti-EBNA-1 antibody and with anti-modified citrulline antibodies. Conclusion. These data indicate that ACPAs react with a viral deiminated protein and suggest that EBV infection may play a role in the induction of these RA-specific antibodies.