Journal of General Virology (2000), 81, 983–992. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Role of Ser-652 and Lys-692 in the protease activity of infectious bursal disease virus VP4 and identification of its substrate cleavage sites Nathalie Lejal, 1 Bruno Da Costa, 1 Jean-Claude Huet 2 and Bernard Delmas 1 Unite  de Virologie et Immunologie mole  culaires 1 and Unite  de Biochimie et Structure des prote  ines 2 , Institut National de la Recherche Agronomique, F-78350 Jouy-en-Josas, France The polyprotein of infectious bursal disease virus (IBDV), an avian birnavirus, is processed by the viral protease, VP4. Previous data obtained on the VP4 of infectious pancreatic necrosis virus (IPNV), a fish birnavirus, and comparative sequence analysis between IBDV and IPNV suggest that VP4 is an unusual eukaryotic serine protease that shares properties with prokaryotic leader peptidases and other bacterial peptidases. IBDV VP4 is predicted to utilize a serine–lysine catalytic dyad. Replacement of the members of the predicted catalytic dyad (Ser-652 and Lys-692) confirmed their indispensability. The two cleavage sites at the pVP2–VP4 and VP4–VP3 junctions were identified by N-terminal sequencing and probed by site-directed mutagenesis. Several additional candidate cleavage sites were identified in the C-terminal domain of pVP2 and tested by cumulative site-directed mutagenesis and expression of the mutant polyproteins. The results suggest that VP4 cleaves multiple (Thr/Ala)–X–Ala  Ala motifs. A trans activity of the VP4 protease of IBDV, and also IPNV VP4 protease, was demonstrated by co-expression of VP4 and a polypeptide substrate in Escherichia coli. For both proteases, cleavage specificity was identical in the cis- and trans-activity assays. An attempt was made to determine whether VP4 proteases of IBDV and IPNV were able to cleave heterologous substrates. In each case, no cleavage was observed with heterologous combinations. These results on the IBDV VP4 confirm and extend our previous characterization of the IPNV VP4, delineating the birnavirus protease as a new type of viral serine protease. Introduction Infectious bursal disease, also named Gumboro disease, is a contagious virus disease of young chickens that is of considerable economical importance in the poultry industry. Infectious bursal disease virus (IBDV) specifically infects B cells present in the bursa of Fabricius. IBDV, like infectious pancreatic necrosis virus (IPNV) and Drosophila X virus (DXV), is a member of the Birnaviridae (Dobos et al., 1995). Birnaviruses contain two double-stranded RNA segments, A and B, within a non-enveloped, single-shelled icosahedral virion, about 65 nm in diameter (Dobos et al., 1979; Bo  ttcher et al., 1997). Segment B (approximately 28 kbp) encodes VP1, a 100 kDa protein with RNA polymerase activity (Morgan et al., 1988). Segment A (approx. 33 kbp) encodes a polyprotein Author for correspondence : Bernard Delmas. Fax 33 1 3465 2621. e-mail delmasbiotec.jouy.inra.fr precursor, which is co-translationally processed into three proteins, pVP2 (also designated VPX), VP3 and VP4 (Hudson et al., 1986). The pVP2 to VP2 conversion involves the cleavage of pVP2 near its C terminus (Azad et al., 1987). VP2 and VP3 are the major structural proteins of virions, whereas VP4 is a virus-encoded protease essential for processing (Azad et al., 1987 ; Jagadish et al., 1988 ; Kibenge et al., 1997), which belongs to the U43 family according to the classification of the MEROPS website (http :www.bi.bbsrc.ac.ukworldLabs peptidaseunkfam.htm). A second open reading frame, par- tially overlapping the 5 polyprotein gene, encodes the non- structural protein VP5 (Mundt et al., 1995). We have recently reported the characterization of the IPNV VP4 protease (Petit et al., 2000), showing that this protease may use a catalytic serine–lysine dyad, a catalytic site previously identified in several prokaryotic peptidases and hydrolases (for a review, see Barrett & Rawlings, 1995). The IPNV VP4 cleavage sites were identified by N-terminal 0001-6763  2000 SGM JID