key: cord-1007808-i5gfaicu
authors: Delmas, Bernard; Mundt, Egbert; Gorbalenya, Alexander E.
title: Chapter 779 Birnavirus VP4 Processing Endopeptidase
date: 2013-12-31
journal: Handbook of Proteolytic Enzymes
DOI: 10.1016/b978-0-12-382219-2.00779-1
sha: ebdad0378c2121e56d06c84697c1e060535456c5
doc_id: 1007808
cord_uid: i5gfaicu

The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 800 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 779 is Birnavirus VP4 Processing Endopeptidase. Keywords Autoproteolytic proteinase release, birnavirus, α/β fold, multifunctional proteinase, narrow substrate specificity, polyprotein processing, RNA virus, replication control, serine-lysine proteinase, virion formation control.

[52] Racchi, M., Watzke, H.H., High, K.A., Lively, M.O. (1993) .

Human coagulation factor X deficiency caused by a mutant signal peptide that blocks cleavage by signal peptidase but not targeting and translocation to the endoplasmic reticulum. J. Biol. Chem. 268 (8) (DBV) and  the Tellina virus 1 (TV-1) , respectively. The virions are nonenveloped, icosahedral particles with a capsid made by the viral protein 2 (VP2, [2] ); the number 2 following 'VP' corresponds to the relative migration position of this protein À from top to bottom À among other virion proteins in SDS-PAGE. The birnavirus genome is made by two dsRNA segments, A and B, each consisting of about 3À3.5 thousand base pairs. Genomic segment A encodes a polyprotein precursor (PP) with the NH 2 -pVP2-VP4-VP3-COOH protein organization; a small protein, called VP5 in IBDV, is encoded in a separate overlapping open reading frame. In BSNV and TV-1, the PP includes an additional polypeptide À called X and flanked by the pVP2 and VP4 À to form the pVP2-X-VP4-VP3 protein organization. Segment B encodes the viral RNA-dependent RNA polymerase (VP1) a fraction of which is covalently bound to the 5 0 -ends of the two strands of the both genomic RNA segments [3] . The birnavirus virus protein 4 processing endopeptidase (VP4) is autoproteolytically released from the PP along with the VP2 precursor pVP2 and VP3 in vivo in infected cells [4À7] and in vitro in cell-free reticulocyte lysates [8, 9] . VP4 remained an enigmatic protease for more than a decade (for a detailed historical account see Leong & Mason [10] ). Two groups have independently presented conclusive evidence for VP4 to be a Ser-Lys protease that is distantly related to bacterial Lon proteases (see Chapters 781 and 782) [8, 9, 11] . VP4 was coined 'non-canonical Lon proteinase' to distinguish it from the bacterial, ATP-dependent homologs [8] . The substrate specificity was first identified for the DXV VP4 protease cleaving between Ser500 and Ala501 and, likely, Ser723 and Ala724 residues (hereafter the PP nomenclature specific for a particular birnavirus) [12] .

The proteolytic activities of IBDV, IPNV, BSNV and TV-1 VP4s were demonstrated in E. coli and reticulocyte lysates [8, 9, 11, 13, 14] and, additionally for the IBDV VP4, in cRNA transfected cells in vivo [8] . The authentic IBDV and IPNV PP proteolytic processing has also been reported in transient expression systems in eukaryotic cells [15À18] . When expressed in these systems, PP or its fragments, containing VP4 with upstream or downstream sequences, were processed to VP4 and (truncated versions of) pVP2, (X), and VP3. The further conversion of pVP2 into VP2 was evident only when virus-like particles were formed, suggesting that the pVP2-to-VP2 proteolytic conversion is linked to virus morphogenesis [19À21] . The IPNV and IBDV VP4s may cleave in cis and trans, although the latter, less efficient activity has been demonstrated only in studies in E. coli and reticulocyte lysates [8, 9, 11] . The PP cleavage at the VP4kVP3 versus the pVP2kVP4 site was found to be more sensitive to point mutations in the IBDV and IPNV VP4 (and not observed in trans for PP formed in reticulocyte lysates). The VP4kVP3 [6, 15] and pVP2kVP4 [5, 22] interactions do not appear to be crucial for the cleavage at the distal site. The proteolytic processing of the IPNV PP was claimed to be resistant to inhibitors of all protease classes, although the details of this analysis was only published in a thesis [10] . Unlike the canonical Lon, VP4 does not depend on ATP to cleave its protein substrate [8] .

VP4 proteases of different birnaviruses were shown to cleave the VP4kVP3 and the pVP2kVP4 (and the pVP2kX and XkVP4) junctions cotranslationally during the PP synthesis. Consequently, PP may never be formed in vivo. Subsequent serial cleavages at the C-terminus of pVP2 yield the mature VP2 protein and, depending on the virus, three (IPNV, TV-1 and DXV) or four (IBDV and BSNV) peptides that remain associated with the virion.

Stepwise conversion of pVP2 to VP2 takes place only upon particle assembly [19À21,23] . It involves a large quaternary maturation complex formed by pVP2 that is processed by VP4 in association to VP3 interacting with VP1 [23] . It seems that a conformation of VP3, which is produced under the VP4 control, may affect the cleavage [19] . The VP4 involvement in the final cleavage leading to the mature VP2 À between VP2 and the largest peptide in IBDV (pep46) À was postulated based upon similarities of this site with the proven VP4 cleavage sites sequences [24] . Alternatively, this cleavage may be VP4independent and requires an Asp431 in VP2 [25] .

The structure of the discussed cleavage sites were identified by different methods including the N-terminal sequencing of the C-terminal cleavage products, sitedirected mutagenesis of these sites, mass spectrometry identification of the virus-associated peptides or their combination. Some cleavage sites in the least characterized birnaviruses were assigned by comparative sequence analysis with experimentally established sites [9,11À14,18,24,26] . Despite the sequence similarities between the VP4 cleavage sites of different birnaviruses, VP4s of IPNV and IBDV were unable to cleave non-cognate substrates indicating the involvement of other species-specific determinants [9] . These and other determinants of substrate specificity were elucidated in the crystal structures of the IPNV and TV-1 VP4s in complex with peptide substrates [27, 28] (see below).

Bioinformatics, molecular genetics and biochemical analyses first identified VP4 as a Ser-Lys catalytic dyad protease containing from 226 to 247 amino acid residues and including an B90 amino acid domain conserved across the Lon/VP4 proteases (core) at its C-terminus [8, 9, 11] . The Ser652 and Lys692 of IBDV and their counterparts in IPNV, BSNV and TV-1 proved to be indispensable for the catalysis. From all mutations tested in the IBDV Ser652 position, the PP processing tolerated only a Ser652Cys replacement [8] . The proteolytic activity of the IBDV Ser652Cys mutant was selectively inactivated by exposure to 3.5 mM N-ethylmaleimide. Likewise, the proteolytic activity of the IBDV Lys692Arg but not four other Lys692 mutants was activated in the monomolecular reaction at pH 11.0. Collectively, these results strongly imply that Lys692 plays the role of a general base and activates the hydroxyl of Ser652 for catalysis [8] . The chemistry of proteolysis used by VP4 is likely to be conserved across the whole Lon protease family.

In contrast to the conservation of the core domain in VP4 and Lon, no statistically significant similarity was detected between the primary structures of VP4 and other proven Ser-Lys proteases. However, the relative position and distance between the catalytic Ser and Lys residues, and results of secondary structure predictions are compatible with the VP4 core adopting a variant of the β-sheet fold conserved in UmuD 0 and leader bacterial protease, prototypes of two other, distantly related, Ser-Lys families (see Chapters 772À778 and this chapter) [8] .

These and other aspects were fully addressed in the crystal structures of the E.coli expressed VP4s of BSNV, IPNV and TV-1 solved by the group of M. Paetzel [27À29] (Figure 779.2) . The VP4s from these three birnaviruses were found to adopt similar protein folds made of two separate domains. Domain I is predominately formed by β-strands mainly arranged in an antiparallel fashion. This β-sheet domain houses the substrate groove and specificity pockets. Domain II adopts an α/β fold, with the nucleophilic serine residing at the amino-terminal end of the first α-helix and the general-base lysine being part of the second helix. The VP4 fold distantly resembles those of other Ser-Lys proteases.

In BSNV, the N ζ of the general base Lys729 is coordinated by the nucleophile Ser692 O γ that together define the catalytic dyad of the protease [29] . The N ζ of Lys729 is also coordinated via hydrogen bonds to the O γ 1 of Thr712 and the O of Pro590. The two catalytic and two catalytic-coordinating residues are universally conserved in all known birnavirus proteases [27] . The accessible surface area of Lys729 N ζ is 5.5 Å as compared to an average of 51 Å for the N ζ of other lysine residues in BSNV VP4. When a cognate cleavage site (a substrate) was modeled into the substrate-binding pocket of VP4, the N ζ of Lys729 was found to be completely buried. This suggests that the ε-amino group of Lys729 can be deprotonated by change in local structural environment that decreases its pK a and enables it to act as a general base during the catalysis.

An important contributor to the catalytic machinery in serine proteases is the oxyanion hole that neutralizes the developing negative charge on the scissile carbonyl oxygen atom of the substrate during the formation of the tetrahedral intermediates. Typically, two main chain amide hydrogen atoms that serve as hydrogen bond donors to the developing oxyanion form oxyanion holes. In the VP4 structure of BSNV without a bound substrate, the main chain NH of the Ser692 and Gln691 residues point towards the binding site; these residues most likely contribute atoms to the oxyanion hole. In contrast, the NH group of the highly conserved Gly690, whose equivalent in many serine proteases forms the oxyanion hole, is pointing away from the binding site. The TV-1 VP4 acylenzyme structure [27] revealed the structural conservation of the oxyanion hole that is formed by the main chain amide nitrogen of the catalytic Ser738 and Asn737 (TV-1 amino acid numbering). Ser738 is within hydrogen bonding distance to the scissile carbonyl oxygen. Because the main chain amide nitrogen of Asn737 is too distant to contribute to the oxyanion stabilization, it is possible that the TV-1 VP4 oxyanion hole is only formed during the transition states when the tetrahedral oxyanion is present.

The acyl-enzyme intermediate complex structure determination of the TV-1 VP4 [27] allowed the identification of a potential 'deacylating' ('nucleophilic') water. It is coordinated by hydrogen bonds to Ser738 O γ , Lys778 N ζ , Pro649 O, and Thr760 O γ 1.

Surface analysis of the BSNV VP4 showed that the largest pocket on the surface of VP4 corresponds to an extended bent crevice that incorporates the catalytic residues at one end [29] . The dimensions of the crevice are consistent with the binding of an extended polypeptide and the topology of the surface suggests that the crevice is the S1/S3 binding pocket. Their sizes are consistent with the size of the side chains at the P1 and P3 positions of the cleavage site. The position of the binding site relative to the nucleophilic serine hydroxyl group shows that Ser692 O γ attacks from the si-face of the scissile bond.

The TV-1 VP4 crystals revealed a continuous electron density from the catalytic Ser738 O γ to the carbonyl carbon of Ala830, the C-terminus of VP4 and P1 residue for the VP4kVP3 cleavage site [27] . Therefore, the structure revealed an intramolecular acyl-enzyme complex with the three last residues of TV-1 VP4 stabilized by hydrogen bonding interactions with the VP4 residues constituting the cleavage-site recognition groove and specificity pockets. Consistent with results obtained for the BSNV VP4, no binding pockets were identified for the P2, P4 and P5 residues whose side chains are pointing away from the substrate-binding site.

The crystal structure of the IPNV VP4 revealed the molecular details of an acyl-enzyme complex formed between the enzyme and an internal VP4 cleavage site of another VP4 molecule in trans [28] . This complex was described using a truncated enzyme in which the general base lysine was substituted by an alanine. In the complex, the nucleophilic Ser633 O γ forms an ester bond with the main chain carbonyl of the C-terminus of a neighboring VP4 molecule in which the internal site is located. The substrate specificity is determined by interactions involving the S1, S3, S5 and S6 substrate-binding pockets and respective residues of the substrate.

Birnaviruses differ in respect to the conservation of VP4 cleavage sites in the PP. Alanine is conserved at positions P1 and P3 of all cleavage sites in TV-1 [14] . In contrast, in IPNV alanine is conserved only at the P1 position while a serine residue is predominantly found at the P3 position of the cleavage sites. Consistently with these patterns, analysis of the molecular surfaces for the S1 and S3 specificity pockets of the TV-1 VP4 revealed that the S1 and S3 are shallow and hydrophobic, complementary of the alanine methyl group side chains at the P1 and P3 positions. Likewise, the IPNV S1 was also shallow and hydrophobic, while the S3 pocket is deeper and hydrophilic, allowing it to adapt with a greater variety of residues at the P3 position. Water molecules at the bottom of the pocket may play a role in the fit for a larger variety of side chains (see Figure 779 .1) [27] . A comparison of the substratebinding grooves with bound substrates revealed a similar hydrogen bonding pattern spanning residues P1 to P5 with an average hydrogen bonding distance of 3.0 Å for both TV-1 and IPNV VP4s [27, 28] . Both substrate-binding grooves utilize a water molecule within the interaction distance, but on the opposite site of the substrate.

Preparation VP4 can be produced as a recombinant protein in prokaryotic and eukaryotic expression systems. Regardless of the virus origin, all VP4s characterized so far were found soluble when expressed in E. coli and amenable for structural and biochemical studies (see Chung & Paetzel [27] and references therein). However, some differences between VP4s from diverse birnaviruses were also revealed. For instance, recombinant expression of the IPNV VP4 results in the generation of several truncated by-products likely produced through the utilization of internal cleavage sites [11] . Consequently, expression of a proteolytically inactive VP4 mutant (with a replacement of the catalytic Ser or Lys residue) yielded a stable fulllength protein. In contrast, wild-type VP4s from BSNV, TV-1 and IBDV have long half-life times and are stable at high concentrations. An additional distinguishing feature of the IBDV VP4 is that it forms tubules made by homo-multimers in infected cells and heterologous expressing systems. These tubules (named type II tubules to differentiate from those formed by pVP2 in infected cells) are 25 nm in diameter. They may change in appearance during isolation [30] and can be recovered by differential centrifugations from E. coli or cellular lysates. Purifications of other recombinant VP4s were mainly carried out using recombinant His-tagged proteins by nickelaffinity and size-exclusion chromatographies.

VP4 regulates the birnavirus replication cycle at several levels. VP4 was detected in IBDV and IPNV virions [22, 31] , although it may be a contamination from unique type II tubules in IBDV [30] . The indispensability of the VP4 proteolytic activity for the virus viability was proved using both IBDV and IPNV ( [8] ; E. Mundt, unpublished data). Further insight into the biological roles of VP4 has been gained using IBDV. The VP4-mediated proteolytic processing of PP is critical for the formation of virion particles, of which two major components, pVP2/VP2 and VP3, are proteolytically derived. Virions of IBDV also contain four peptides derived upon the conversion of pVP2 into VP2. Two IBDV mutants, in which a locus encoding either the first or last VP2 peptide was deleted, were found to be noninfectious [24] . When the PP processing was blocked or heavily impaired by a mutation of the active site of VP4, the expression of VP1 encoded by RNA segment B was not evident in transfected cells [8] . VP4 might trans activate the expression of RNA B (VP1) through the production of VP3 that is known to interact with VP1 [23, 32, 33] and may bind virus RNAs [19, 34] . Complete but slowed PP processing in cells transfected by the active site Ser652Cys mutant was apparently not sufficient for the generation of infectious progeny, indicating that virion biogenesis may be coupled with the PP cleavages in a cleavage-rate restricted manner [8] .

The birnavirus VP4s form a unique branch of the Lon protease family, which protease domain has a narrow substrate specificity and neither needs ATP for its activity nor contains an ATPase domain. Proteases with a similar domain organization are encoded by eukaryotes [8] .

The property of the IBDV VP4 to form tubules with a diameter of 25 nm in vivo is not shared with the VP4 of other birnaviruses. Mouse monoclonal antibodies directed against VP4 of IBDV strain P2 were described [30] .

For related peptidases see Chapters 781 and 782 on Lon proteases, and a separate chapter (780) on TV-1 VP4 peptidase.

The papers of Birghan et al. [8] , Lejal et al. [9] , Petit et al. [11] , da Costa et al. [24] , Chung & Paetzel [27] , Lee et al. [28] , and Feldman et al. [29] are recommended.

Virus Taxonomy, Nineth Report of the International Committee on Taxonomy of Viruses

The birnavirus crystal structure reveals structural relationships among icosahedral viruses

Conversion of VP1 to VPg in cells infected by infectious pancreatic necrosis virus

Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus

Synthesis of the infectious pancreatic necrosis virus polyprotein, detection of a virus-encoded protease, and fine structure mapping of genome segment A coding regions

Birnavirus precursor polyprotein is processed in Escherichia coli by its own virus-encoded polypeptide

Cell-free translational analysis of the processing of infectious pancreatic necrosis virus polyprotein

A non-canonical lon proteinase lacking the ATPase domain employs the Ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus

Role of Ser-652 and Lys-692 in the protease activity of infectious bursal disease virus VP4 and identification of its substrate cleavage sites

Infectious pancreatic necrosis virus endopeptidase

Active residues and viral substrate cleavage sites of the protease of the birnavirus infectious pancreatic necrosis virus

Sequence analysis of the bicistronic Drosophila X virus genome segment A and its encoded polypeptides

Blotched snakehead virus is a new aquatic birnavirus that is slightly more related to avibirnavirus than to aquabirnavirus

Genome and polypeptides characterization of Tellina virus 1 reveals a fifth genetic cluster in the Birnaviridae family

Infectious bursal disease virus polyprotein processing does not involve cellular proteases

Formation of virus-like particles when the polyprotein gene (segment A) of infectious bursal disease virus is expressed in insect cells

Expression of ORF A1 of infectious bursal disease virus results in the formation of virus-like particles

Proteolytic processing in infectious bursal disease virus: identification of the polyprotein cleavage sites by site-directed mutagenesis

The maturation process of pVP2 requires assembly of infectious bursal disease virus capsids

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus

Processing of infectious bursal disease virus (IBDV) polyprotein and self-assembly of IBDV-like particles in Hi-5 cells

Evidence for the detection of the infectious pancreatic necrosis virus polyprotein and the 17-kDa polypeptide in infected cells and of the NS protease in purified virus

Structural peptides of a nonenveloped virus are involved in assembly and membrane translocation

The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2

Autoproteolytic activity derived from the infectious bursal disease virus capsid protein

Peptides resulting from the pVP2 C-terminal processing are present in infectious pancreatic necrosis virus particles

Crystal structure of a viral protease intramolecular acyl-enzyme complex: insights into ciscleavage at the VP4/VP3 junction of Tellina birnavirus

Crystal structure of the VP4 protease from infectious pancreatic necrosis virus reveals the acyl-enzyme complex for an intermolecular selfcleavage reaction

Crystal structure of a novel viral protease with a serine/lysine catalytic dyad mechanism

A second form of infectious bursal disease virusassociated tubule contains VP4

Biochemistry and immunology of infectious bursal disease virus

VPl, the putative RNA-dependent RNA polymerase of infectious bursal disease virus, forms complexes with the Capsid Protein VP3, leading to efficient encapsidation into virus-like particles

Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy

Sequence Logos À a new way to display consensus sequences

Email: Bernard.delmas@jouy.inra.fr Egbert Mundt Department of Population Health, Poultry Diagnostic and Research Center

LUMC E4-P, PO Box 9600, 2300 RC Leiden, The Netherlands. Email: a.e.gorbalenya@lumc.nl Handbook of Proteolytic Enzymes, 3rd Edn ©