Abstract
Infectious bursal disease virus (IBDV) is a member of the birnavirus family and is the causative agent of infectious bursal disease (IBD). The virus has a bisegmented dsRNA genome; the larger of these 2 segments is segment A and has 3 open reading frames (ORFs). One of these ORFs encodes a polyprotein that is processed to 3 polypeptides VPX, VP3, and VP4. VP4 has been demonstrated to be responsible for the cleavage of the polyprotein in an autocatalytic co-translational manner. The VP4 protein has not yet been characterised and has no homology with any other known protease except a limited homology with the Lon protease in bacteria. Several active sites are proposed; with the spacing of type I serine proteases, with spacing of type II serine proteases and with spacing of the beta-lactamase family of proteases. The cleavage sites have been proposed by molecular weight determination as being 2 dibasic residues at either end of VP4. Here I describe the expression of the full length polyprotein in a coupled transcription/translation system and in E. coli. Deletion mutagenesis of the VP4 revealed that VP4 is required for correct processing of the processing of VP3 and the processing of VPX to VP2. However, the processing of VP3 is only completely abolished when a deletion into VP3 passed the A-x-AAS cleavage site. Deletion mutagenesis of the N terminus of VP3 in the polyprotein showed that the cleavage sites of VP4 as being A-x-AAS sequences. Site directed mutagenesis studies within VP4 revealed several important residues for the correct processing, the most important of these being the S652. Of the residues mutated here, there does not seem to be a "classical" catalytic triad for this protease.