Characterization of the RNA-binding activity of VP3, a major structural protein of Infectious bursal disease virus.
Infectious bursal disease virus (IBDV) is the agent of an immune-depressive disease affecting the poultry industry worldwide. Infection of IBDV leads to expression of five mature virus-encoded proteins. Proteolytic processing of the virus-encoded polyprotein generates VP3 which coats the inner surface of the IBDV capsid. In this report, we describe the characterization of the RNA-binding activity of VP3. For these studies, the VP3 coding region was fused to a histidine tag and expressed in insect cells using a recombinant baculovirus. The histidine-tagged VP3 was affinity-purified and used to study its ability to bind RNA molecules using three complementary methods: (i) Northwestern blotting; (ii) binding of VP3 protein-RNA complexes to nitrocellulose membranes; and (iii) electrophoretic mobility shift assays. The results demonstrated that VP3 efficiently bound ssRNA and dsRNA. Under the experimental conditions used in this study, the formation of VP3-RNA complexes did not depend upon the presence of specific RNA sequences. A series of histidine-tagged VP3 deletion mutants spanning the whole VP3 coding region were generated. The use of these mutants revealed that the VP3 RNA-binding domain layed in a highly conserved 69 aa stretch close to the N-terminus of the protein.
Kochan
G
G
Departamento de Biología Molecular y Celular, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Madrid, Spain.
Gonzalez
D
D
Rodriguez
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D007243:Infectious bursal disease virus / Q000378:metabolism*; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013045:Species Specificity
D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins