Antibodies to the Epstein-Barr virus nuclear antigen and to rheumatoid arthritis nuclear antigen identify the same polypeptide.

Abstract

Patients with seropositive rheumatoid arthritis (RA) have elevated titers of precipitating antibody toward an antigen designated RA nuclear antigen (RANA). Anti-RANA reactivity has been associated with prior Epstein-Barr virus (EBV) be reactive when a complement fixation assay was used in the immunoblot. Finally, when the Mr 80,000 antigen was used to absorb an anti-RANA/anti-EBNA serum, both antibodies were reduced.The rheumatoid arthritis nuclear antigen (RANA) was first identified in WI-L2 cells by a precipitating antibody seen in the sera of most rheumatoid arthritis (RA) patients but uncommonly in normal sera (1, 2). RANA was shown to be a nuclear antigen expressed exclusively in Epstein-Barr virus (EBV) genome-positive B-lymphoblast lines (3). With more sensitive assays, anti-RANA can now be detected in many normal sera, although in lower titer than in RA sera (4-7), and also during recovery from acute infectious mononucleosis (IM) (5).The relation between RANA and the Epstein-Barr nuclear antigen (EBNA) (8) has remained a matter of some uncertainty. RANA was originally detected in extracts of EBV-infected cells by precipitation reactions but has also been detected in an assay using heat fixation and anti-IgG immunofluorescence (IF) (1, 2). EBNA was originally assayed on acetone-fixed cells by anticomplement immunofluorescence (ACIF) and later by complement fixation with soluble cell extracts (8, 9). EBNA staining typically displays a plaque-like or diffuse stain confined to the nucleus (8), whereas RANA is more granular or speckled and sometimes extends into the cytoplasm (2). RANA and EBNA staining patterns are maximally expressed at different times in the cell cycle of WI-L2 cells and they are differentially expressed in somatic cell hybrids (10, 11). Significant differences in anti-EBNA and anti-RANA titers have been reported for individual sera-notably, those from Burkitt lymphoma patients (3). However, antibodies reactive with EBNA and RANA have been shown to arise roughly in parallel after EBV infection (5), and anti-EBNA'and anti-RANA titers in individual RA patients and normal adults are closely correlated (4, 6). Only EBNA has been partially purified and characterized; however, the EBNA sizes reported range from Mr 48,000 to Mr 100,000 (12-15 Nuclear Isolation and Extraction. After washing in phosphate-buffered saline (150 mM NaCl/10 mM sodium phosphate, pH 7.4) containing 0.2 mM phenylmethylsulfonyl fluoride, cells were swollen for 5 min in reticulocyte standard buffer (RSB; 10 mM NaCl/10 mM Tris-HCl, pH 7.4/1.5 mM MgCl2/ 0.2 mM phenylmethylsulfonyl fluoride). The cells were pelleted and homogenized in RSB containing 0.2% Nonidet P-40; then nuclei were sonicated in 3-5 vol of the same buffer adjusted to 0.2-0.35 M NaCl. After 30 min on ice, the nuclear extract was centrifuged at 10,000 x g and used as a source of antigen. When heat fractionation was used, the extract was diluted to ca. 0.15 M NaCl and rapidly heated to 750C, held for 10 min, chilled, and centrifuged at 20,000 X g for 1...

Correlation between the presence of antibodies to the Epstein‐barr virus nuclear antigen type 2 and antibodies to the rheumatoid arthritis nuclear antigen in patients with rheumatoid arthritis

“…We found that inclusion of SDS in the transfer buffer was required to obtain efficient transfer of EBNA-2 onto nitrocellulose, and that the binding of EBNA-2 to the nitrocellulose was not stable since overnight incubation of the nitrocellulose in saline resulted in the loss of a significant amount of the antigen. Billings et a1 (10) did not include SDS in their transfer buffer, and this is the likely reason for the discrepancy between results.…”

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

“…EBV infection is widespread, and 95% of all adults display serologic signs of a previous infection. It is known that patients with RA have elevated levels of antibodies to latent and replicative EBV proteins (36) and in particular to EBNA-1 (37,38).…”

Synovial Epstein-Barr virus infection increases the risk of rheumatoid arthritis in individuals with the shared HLA-DR4 epitope

“…Among several viruses that have been implicated in the pathogenesis of RA (6)(7)(8), a possible relationship between Epstein-Barr virus (EBV) and RA was initially suspected from disease-specific, altered humoral immune responses toward different viral antigens, such as the Epstein-Barr nuclear antigens (EBNAs) 1 and 2, in patients with RA (9)(10)(11)(12). Antibodies against a glycine-alanine repeat in the EBNA-1 molecule were shown to cross-react with a 62-kd protein present in the synovial tissues of patients with RA but absent from the joints of patients with osteoarthritis (OA) and from normal subjects (13).…”