Structural and Mutational Analysis of an Antibody to the Epstein Barr Virus Nuclear Antigen-1 (EBNA-1) that Cross-reacts with dsDNA

The Epstein Barr Virus (EBV) has been linked to the autoimmune disease systemic lupus erythematosus (SLE). SLE is marked by the presence of pathogenic IgG antibodies to double stranded DNA (dsDNA). How these autoantibodies arise is unknown but molecular mimicry between an EBV nuclear protein (EBNA-1) and dsDNA may play a role. We previously generated a monoclonal antibody (3D4) to EBNA-1 and demonstrated that it cross-reacts with dsDNA. This antibody recognizes an epitope in the carboxyl region of EBNA-1 (EBNA-1_459-619) and has pathogenic potential. We have crystallized 3D4 complexed to EBNA-1_(459-619) and identified residues in the CDR regions that contact EBNA-1_(459-619). We have mutated these contact residues in 3D4 and demonstrated that mutations in CDR1 and CDR2 of the light chain (LC) diminish binding to EBNA-1_(459-619) and dsDNA. Mutations in CDR1 of the heavy chain (HC) do not alter binding to EBNA-1_(459-619) or dsDNA, however, mutations in CDR2 HC abolish binding to dsDNA only, while mutations in CDR3 HC abolish binding to both. Our data indicate that the CDR3 HC is necessary for binding EBNA-1 and dsDNA but that CDR2 is only necessary for binding to dsDNA and confirms the bi-specificity of the antibody.  These results also support our hypothesis that EBNA-1 can structurally mimic dsDNA and thereby elicit cross-reactive antibodies. Understanding the basis for this structural cross-reactivity may have future diagnostic and therapeutic significance.