Direct investigation of protein RNA-binding domains using digoxigenin-labelled RNAs and synthetic peptides: application to the hepatitis delta antigen.
A new method is described for the characterization of RNA binding domains of a protein and applied to the study of the interaction between proteins 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 quantified with peroxidase-conjugated anti-digoxigenin. The hepatitis delta antigen (HDAg) is an RNA-binding protein that specifically binds HDV RNAs. In a previous study, it was shown that HDAg sequences corresponding to residues 2-27 and 79-107 bound to both genomic and antigenomic strands. Further investigations are reported on HDAg/HDV RNA binding, using additional HDAg peptides and the full-length HDV genomic and antigenomic strands. In order to validate the method, the efficiency of peptide coating onto the ELISA plate was assessed with human antibodies against HDAg. The two arginine-rich motifs potentially involved in the RNA-binding activity (97-107 and 136-146) were explored and the residues 2-27 and 79-211 were mapped using synthetic peptides. Only peptides corresponding to residues 2-17, 2-27, 79-107 and 84-126 of the HDAg 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 involved in this interaction without a direct ability to bind HDV RNAs.
Poisson
F
F
Département de Microbiologie Médicale et Moléculaire, URA CNRS 1334, CHU Bretonneau, Tours, France.
Roingeard
P
P
Goudeau
A
A
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D004076:Digoxigenin / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018963:Hepatitis Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003698:Hepatitis Delta Virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D035923:Hepatitis delta Antigens; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
D018963:Hepatitis Antigens; D035923:Hepatitis delta Antigens; D010455:Peptides; D012367:RNA, Viral; D016601:RNA-Binding Proteins; C442244:hepatitis delta virus large antigen; D001120:Arginine; D004076:Digoxigenin