Active Residues and Viral Substrate Cleavage Sites of the Protease of the Birnavirus Infectious Pancreatic Necrosis Virus

Abstract

The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and Drosophila X virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala2(Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.

Novel Predicted Peptidases with a Potential Role in the Ubiquitin Signaling Pathway

“…This is a new contribution to the diversity of (predicted) proteases of eukaryotic viruses. These include distinct superfamilies of the papain-like thiol proteases, at least two groups of chymotrypsin-like thiol and serine proteases, 43 the LON-type proteases, 56 the NlpC-like peptidases, 29 the OTU-like proteases, 13 and, as shown here, the PPPDE-like predicted protease. At least five families of viral proteases are specifically related to known or predicted eukaryotic DUBs, namely, picornavirus papain-like proteases derived from UCH-like cysteine proteases, the OTU-like proteases of arteriviruses and some dsRNA viruses, the PPPDE-like predicted proteases of hypoviruses and reoviruses, the Smt4p-like proteases of adenoviruses, poxviruses, and other nucleo-cytoplasmic large dsDNA viruses, 48,57,58 and the Ddi1p-like aspartyl proteases of retroviruses.…”

Molecular characterisation of Australasian isolates of aquatic birnaviruses

“…If the polyprotein cleavage sites were assumed to be (Petit et al 2000), the processed proteins VP2, NS and VP3 would be 508, 225 and 139 aa in length, respectively. Across the entire polyprotein, TAB98 showed the highest identity with IPNV strain Sp (91% nt, 96% aa) and the lowest identity with IPNV strain He (75% nt, 85% aa).…”

Molecular characterization of Sp serotype strains of infectious pancreatic necrosis virus exhibiting differences in virulence

“…Segment A (3097 bp) encodes a 106 kDa precursor protein in a single large open reading frame (ORF) that is cotranslationally cleaved by the viral nonstructural (NS) protease, VP4, to generate mature VP2 and VP3 structural proteins (Duncan & Dobos 1986, Duncan et al 1987. Precise cleavage sites between the VP2-VP4 and VP4-VP3 junctions of the polyprotein have been identified (Petit et al 2000). In addition, Segment A encodes a 17 kDa arginine-rich NS protein from a small ORF (also known as VP5), which precedes and partially overlaps the polyprotein ORF (Magyar & Dobos 1994).…”