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 immunedepressive 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.