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-Ala / (Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.

FIG. 1

Patterns of conserved amino acids in birnavirus VP4s. Arrows indicate approximate positions of the cleavage sites. Three regions, A to C, containing the most conserved patterns of amino acids, are shown. The comparison includes the deduced amino acid sequences of VP4 of IPNV strain SP (accession number U56907), IBDV (accession number P15480), and DSX (accession number U60650). Identical residues are in boldface when present at least in two sequences. Numbers refer to the position of the first amino acid of each domain in the respective VP4s. Residues proposed to be part of the catalytic site of VP4s (•) are indicated.

FIG. 2

Mutagenesis of the conserved histidine, aspartic acid, and glutamic acid residues of the IPNV VP4. The autoradiographs show the results obtained with SKΔIPNA wild type (wt) and a set of mutant SKΔIPNA-derived constructs encoding proteins with a single (or double) amino acid substitution(s). (A) Substitution of histidine residues. (B) Substitution of aspartic acid and glutamic acid residues. The constructs were expressed with the rabbit reticulocyte expression system (Promega), and expression products were analyzed by SDS-PAGE. p2, pVP2; 3, VP3; 4, VP4; 4-3, VP4-VP3; p2-4-3, uncleaved polyprotein precursor. Note that single mutations can strongly affect the behavior of the VP4 in SDS-PAGE.

FIG. 3

Mutagenesis of the conserved serine residues in the B region of IPNV VP4. A number of substitutions were introduced at the position of serines 633 and 639. Expression was carried out as described for Fig. 2.

FIG. 4

Mutagenesis of conserved residues in the regions A, B, and C of VP4. The autoradiographs show the results obtained with SKΔIPNA wild type (wt) and a set of mutant SKΔIPNA-derived constructs encoding proteins with a single amino acid substitution. Region A, left panel; region B, middle panel; region C, right panel. Expression was carried out as described for Fig. 2.

FIG. 5

Mutagenesis of the lysine 674 of IPNV VP4. A number of substitutions were introduced at the position of lysine 674. Expression was carried out as described for Fig. 2.

FIG. 6

Mutagenesis of hydrophobic residues in the region C of the IPNV VP4. Expression was carried out as described for Fig. 2.

FIG. 7

Mapping of the cleavage site at the VP4-VP3 junction. (A) Scheme of the set of three 5′-truncated expression products. Numbers refer to the amino acid position on the full-length ORF polyprotein, and additional sequences are indicated in italics. The HisTag-T7Tag (NH₂ extremity) and the HisTag (COOH extremity), which derive from pET-28 vector, are made of 33 residues and 21 residues, respectively. Mutated residues are indicated in boldface. (B) Expression in reticulocyte lysates of the polyprotein mutant carrying a double substitution at the P1-P′1 position of the cleavage site between the VP4 and VP3 proteins. 3*, 34-kDa band; 4*, 27-kDa band.

FIG. 8

Mapping of the cleavage site at the pVP2-VP4 junction. (A) Schematic representation of the set of 5′-truncated expression products. Numbers refer to the amino acid position on the full-length ORF polyprotein, and additional sequences indicated in italics are as described in Fig. 8. (B) Expression was carried out as described for Fig. 2. (C) Mutagenesis of the cleavage site. Expression in reticulocyte lysates of polyprotein mutants carrying double (at the P1-P′1 position) or single (at the P1, P′1, and P′2 positions) substitutions of the cleavage site between the pVP2 and VP4 proteins. 2*, short pVP2.

FIG. 9

Cleavage sites (and putative cleavage sites) for the IPNV VP4 in the polyprotein. (Top) Schematic representation of the IPNA polyprotein. The amino acids of the VP2-VP4 and VP4-VP3 domains are indicated. The P1-P′1 cleavage site positions are underlined. (Bottom) The sequences relative to cleavage sites identified by N-terminal sequencing are in boldface. The sequences in regular typeface were identified by sequence homology and probed by mutagenesis.

FIG. 10

Mutagenesis of potential cleavage sites for the VP4 protease in the COOH part of pVP2. Expression in reticulocyte lysates of the polyprotein mutants carrying double substitution(s) at the P1-P′1 position of the potential cleavage sites (positions 486 and 487 and positions 495 and 496). p2-4, VP2-VP4; 2∗, short pVP2.