DIRECT INVESTIGATION OF PROTEIN RNA-BINDING DOMAINS USING DIGOXIGENIN-LABELED RNAS AND SYNTHETIC PEPTIDES - APPLICATION TO THE HEPATITIS-DELTA ANTIGEN

A new method is described for the characterization of RNA binding doma ins of a protein and applied to the study of the interaction between p roteins and nucleic acid of the human hepatitis delta virus (HDV). The method uses synthetic peptides coated onto an ELISA plate and tested for their ability to bind digoxigenin-labelled RNAs. RNA binding is qu antified with peroxidase-conjugated anti-digoxigenin. The hepatitis de lta antigen (HDAg) is an RNA-binding protein that specifically binds H DV RNAs. In a previous study, it was shown that HDAg sequences corresp onding to residues 2-27 and 79-107 bound to both genomic and antigenom ic strands. Further investigations are reported on HDAg/HDV RNA bindin g, using additional HDAg peptides and the full-length HDV genomic and antigenomic strands. In order to validate the method, the efficiency o f peptide coating onto the ELISA plate was assessed with human antibod ies against HDAg. The two arginine-rich motifs potentially involved in the RNA-binding activity (97-107 and 136-146) were explored and the r esidues 2-27 and 79-211 were mapped using synthetic peptides. Only pep tides corresponding to residues 2-17, 2-27, 79-107 and 84-126 of the H DAg bound to the genomic and antigenomic strands. The second arginine- rich motif represented by peptides 130-144 and 128-152 did not bind to HDV RNAs in this assay. This second arginine-rich domain may be invol ved in this interaction without a direct ability to bind HDV RNAs.