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

The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.     

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

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.     

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

Infectious bursal disease virus (IBDV) is a nonenveloped avian virus with a two-segment double-stranded RNA genome. Its T=13 icosahedral capsid is most probably assembled with 780 subunits of VP2 and 600 copies of VP3 and has a diameter of about 60 nm. VP1, the RNA-dependent RNA polymerase, resides inside the viral particle. Using a baculovirus expression system, we first observed that expression of the pVP2-VP4-VP3 polyprotein encoded by the genomic segment IBDA results mainly in the formation of tubules with a diameter of about 50 nm and composed of pVP2, the precursor of VP2. Very few virus-like particles (VLPs) and VP4 tubules with a diameter of about 25 nm were also identified. The inefficiency of VLP assembly was further investigated by expression of additional IBDA-derived constructs. Expression of pVP2 without any other polyprotein components results in the formation of isometric particles with a diameter of about 30 nm. VLPs were observed mainly when a large exogeneous polypeptide sequence (the green fluorescent protein sequence) was fused to the VP3 C-terminal domain. Large numbers of VLPs were visualized by electron microscopy, and single particles were shown to be fluorescent by standard and confocal microscopy analysis. Moreover, the final maturation process converting pVP2 into the VP2 mature form was observed on generated VLPs. We therefore conclude that the correct scaffolding of the VP3 can be artificially induced to promote the formation of VLPs and that the final processing of pVP2 to VP2 is controlled by this particular assembly. To our knowledge, this is the first report of the engineering of a morphogenesis switch to control a particular type of capsid protein assembly.     

Host proteolytic activity is necessary for infectious bursal disease virus capsid protein assembly

In many viruses, a precursor particle, or procapsid, is assembled and undergoes massive chemical and physical modification to produce the infectious capsid. Capsid assembly and maturation are finely tuned processes in which viral and host factors participate. We show that the precursor of the VP2 capsid protein (pVP2) of the infectious bursal disease virus (IBDV), a double-stranded RNA virus, is processed at the C-terminal domain (CTD) by a host protease, the puromycin-sensitive aminopeptidase (PurSA). The pVP2 CTD (71 residues) has an important role in determining the various conformations of VP2 (441 residues) that build the T = 13 complex capsid. pVP2 CTD activity is controlled by co- and posttranslational proteolytic modifications of different targets by the VP4 viral protease and by VP2 itself to yield the mature VP2-441 species. Puromycin-sensitive aminopeptidase is responsible for the peptidase activity that cleaves the Arg-452-Arg-453 bond to generate the intermediate pVP2-452 polypeptide. A pVP2 R453A substitution abrogates PurSA activity. We used a baculovirus-based system to express the IBDV polyprotein in insect cells and found inefficient formation of virus-like particles similar to IBDV virions, which correlates with the absence of puromycin-sensitive aminopeptidase in these cells. Virus-like particle assembly was nonetheless rescued efficiently by coexpression of chicken PurSA or pVP2-452 protein. Silencing or pharmacological inhibition of puromycin-sensitive aminopeptidase activity in cell lines permissive for IBDV replication caused a major blockade in assembly and/or maturation of infectious IBDV particles, as virus yields were reduced markedly. PurSA activity is thus essential for IBDV replication.     

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

Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH(2)-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-A resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser(633)Ogamma forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala(716), of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-A resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity ( approximately 19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.     

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

The capsid proteins VP2 and VP3 of infectious bursal disease virus, a birnavirus, are derived from the processing of a large polyprotein: NH2-pVP2-VP4-VP3-COOH. Although the primary cleavage sites at the pVP2-VP4 and VP4-VP3 junctions have been identified, the proteolytic cascade involved in the processing of this polyprotein is not yet fully understood, particularly the maturation of pVP2. By using different approaches, we showed that the processing of pVP2 (residues 1 to 512) generated VP2 and four small peptides (residues 442 to 487, 488 to 494, 495 to 501, and 502 to 512). We also showed that in addition to VP2, at least three of these peptides (residues 442 to 487, 488 to 494, and 502 to 512) were associated with the viral particles. The importance of the small peptides in the virus cycle was assessed by reverse genetics. Our results showed that the mutants lacking the two smaller peptides were viable, although the virus growth was affected. In contrast, deletions of the domain 442 to 487 or 502 to 512 did not allow virus recovery. Several amino acids of the peptide 502 to 512 appeared essential for virus viability. Substitutions of the P1 and/or P1" position were engineered at each of the cleavage sites (P1-P1": 441-442, 487-488, 494-495, 501-502, and 512-513). Most substitutions at the pVP2-VP4 junction (512-513) and at the final VP2 maturation cleavage site (441-442) were lethal. Mutations of intermediate cleavage sites (487-488, 494-495, and 501-502) led to viable viruses showing different but efficient pVP2 processing. Our data suggested that while peptides 488 to 494 and 495 to 501 play an accessory role, peptides 442 to 487 and 502 to 512 have an unknown but important function within the virus cycle.     

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

By different approaches, we characterized the birnavirus blotched snakehead virus (BSNV). The sequence of genomic segment A revealed the presence of two open reading frames (ORFs): a large ORF with a 3,207-bp-long nucleotide sequence and a 417-nucleotide-long small ORF located within the N-terminal half of the large ORF, but in a different reading frame. The large ORF was found to encode a polyprotein cotranslationally processed by the viral protease VP4 to generate pVP2 (the VP2 precursor), a 71-amino-acid-long peptide ([X]), VP4, and VP3. The two cleavage sites at the [X]-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing. We showed that the processing of pVP2 generated VP2 and several small peptides (amino acids [aa] 418 to 460, 461 to 467, 468 to 474, and 475 to 486). Two of these peptides (aa 418 to 460 and 475 to 486) were positively identified in the viral particles with 10 additional peptides derived from further processing of the peptide aa 418 to 460. The results suggest that VP4 cleaves multiple Pro-X-Ala downward arrow Ala motifs, with the notable exception of the VP4-VP3 junction. Replacement of the members of the predicted VP4 catalytic dyad (Ser-692 and Lys-729) confirmed their indispensability in the polyprotein processing. The genomic segment B sequence revealed a single large ORF encoding a putative polymerase, VP1. Our results demonstrate that BSNV should be considered a new aquatic birnavirus species, slightly more related to IBDV than to IPNV.     

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.     

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

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.     

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

The capsid of infectious bursal disease virus (IBDV), with a size of 60-65 nm, is formed by an initial processing of polyprotein (pVP2-VP4-VP3) by VP4, subsequent assemblage of pVP2 and VP3, and the maturation of VP2. In Sf9 cells, the processing of polyprotein expressed was restrained in the stage of VP2 maturation, leading to a limited production of capsid, i.e., IBDV-like particles (VLPs). In the present study, another insect cell line, High-Five (Hi-5) cells, was demonstrated to efficiently produce VLPs. Meanwhile, in this system, polyprotein was processed to pVP2 and VP3 protein and pVP2 was further processed to the matured form of VP2. Consequently, Hi-5 cells are better in terms of polyprotein processing and formation of VLPs than Sf9. In addition to the processing of pVP2, VP3 was also degraded. With insufficient intact VP3 protein present for the formation of VLPs, the excessive VP2 form subviral particles (SVPs) with a size of about 25 nm. The ratio of VLPs to SVPs is dependent on the multiplicity of infections (MOIs) used, and an optimal MOI is found for the production of both particles. VLPs were separated from SVPs with a combination of ultracentrifugation and gel-filtration chromatography, and a large number of purified particles of both were obtained. In conclusion, the insect cell lines and MOIs were optimized for the production of VLPs, and pure VLPs with morphology similar to that of the wild-type viruses can be effectively prepared. The efficient production and purification of VLPs benefits not only the development of an antiviral vaccine against IBDV but also the understanding of the structure of this avian virus that is economically important.     

Avibirnavirus VP4 Protein Is a Phosphoprotein and Partially Contributes to the Cleavage of Intermediate Precursor VP4-VP3 Polyprotein

Birnavirus-encoded viral protein 4 (VP4) utilizes a Ser/Lys catalytic dyad mechanism to process polyprotein. Here three phosphorylated amino acid residues Ser538, Tyr611 and Thr674 within the VP4 protein of the infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus of the family Birnaviridae, were identified by mass spectrometry. Anti-VP4 monoclonal antibodies finely mapping to phosphorylated (p)Ser538 and the epitope motif ⁵³⁰PVVDGIL⁵³⁶ were generated and verified. Proteomic analysis showed that in IBDV-infected cells the VP4 was distributed mainly in the cytoskeletal fraction and existed with different isoelectric points and several phosphorylation modifications. Phosphorylation of VP4 did not influence the aggregation of VP4 molecules. The proteolytic activity analysis verified that the pTyr611 and pThr674 sites within VP4 are involved in the cleavage of viral intermediate precursor VP4-VP3. This study demonstrates that IBDV-encoded VP4 protein is a unique phosphoprotein and that phosphorylation of Tyr611 and Thr674 of VP4 affects its serine-protease activity.     

Interaction mechanism of marine birnavirus (MABV) in fish cell lines

Marine birnavirus (MABV) is a member of the genus Aquabirnavirus of the family Birnaviridae. MABV is an unenveloped icosahedral virus about 60 nm in diameter with two genomes of double-stranded RNA. MABV adsorbed not only onto the cell surfaces of susceptible (CHSE-214 and RSBK-2) cells but also onto resistant (FHM and EPC) cells. Furthermore, the virus entered into the cytoplasm through the endocytotic pathway in CHSE-214, RSBK-2 and FHM cells but did not penetrate EPC cells. The virus was found to bind to an around 250 kDa protein on CHSE-214, RSBK-2, FHM and EPC cells. The syntheses of viral proteins pVP2, NS and VP3 and further proteolytic processing after viral infection were examined by using Western blot analysis. pVP2, NS and VP3 were detected in the cytosolic fractions of CHSE-214, RSBK-2 and FHM cells at 4 h after infection. At this time, VP3 underwent further proteolytic processing in the cytosolic fractions of CHSE-214 and RSBK-2 cells. The expression of pVP2, NS and VP3 increased and pVP2 and NS also underwent further proteolytic processing similar to VP3 in the cytosolic fractions of CHSE-214, RSBK-2 and FHM cells at 8 h after infection. The further proteolytic processing of VP3 was detected in the nuclear fractions of CHSE-214, RSBK-2, but VP3 was detected as a single band in the nuclear fraction of FHM cells. pVP2 and NS were detected as thin bands only in the nuclear fractions of CHSE-214 cells. The results of Western blot analysis demonstrated that pVP2, NS and VP3 are localized in the nuclear fraction when they were independently expressed in CHSE-214, RSBK-2, FHM and EPC cells. The expression pattern in the cytosolic fraction was identical among the four cell lines when pVP2 and NS were independently expressed. However, pVP2 and NS were not detected in the nuclear fraction of CHSE-214 cells. Further proteolytic processing of VP3 was detected in both cytosolic and nuclear fractions of RSBK-2 ,FHM and EPC cells (Low level in EPC cell), but not in CHSE-214 cells when VP3 was independently expressed. Then, the processes of preVP2 to form morphological assemblages in the presence of VP3 or the cleavage of VP3 into two proteins in CHSE-214 cells were studied. When preVP2- and VP3 were co-expressed, virion like particles (64 nm, diameter) were observed close to the nuclear membrane by electron microscopy. The co-expression of preVP2 and the cleaved VP3 proteins led to an efficient assembly of tubules (22 nm, diameter). Further important finds will be obtained by this infection system using 4 fish cell lines in the next couple of years.     

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

The cDNA fragment of the large RNA segment of infectious bursal disease virus 002-73, when expressed in Escherichia coli, produces precursor polyprotein (N-VP2-VP4-VP3-C), most of which is then processed to generate constituent polypeptides. Using cDNA fragments containing site-specific mutations and two monoclonal antibodies that are specific to VP2 and VP3 of mature virus particles, we demonstrated that the VP4 protein is involved in processing of the precursor polyprotein to generate VP2 and VP3 and excluded the possibility of internal initiation for the generation of VP3.     

Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.     

Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch

Infectious bursal disease virus (IBDV), a double-stranded RNA (dsRNA) virus belonging to the Birnaviridae family, is an economically important avian pathogen. The IBDV capsid is based on a single-shelled T=13 lattice, and the only structural subunits are VP2 trimers. During capsid assembly, VP2 is synthesized as a protein precursor, called pVP2, whose 71-residue C-terminal end is proteolytically processed. The conformational flexibility of pVP2 is due to an amphipathic alpha-helix located at its C-terminal end. VP3, the other IBDV major structural protein that accomplishes numerous roles during the viral cycle, acts as a scaffolding protein required for assembly control. Here we address the molecular mechanism that defines the multimeric state of the capsid protein as hexamers or pentamers. We used a combination of three-dimensional cryo-electron microscopy maps at or close to subnanometer resolution with atomic models. Our studies suggest that the key polypeptide element, the C-terminal amphipathic alpha-helix, which acts as a transient conformational switch, is bound to the flexible VP2 C-terminal end. In addition, capsid protein oligomerization is also controlled by the progressive trimming of its C-terminal domain. The coordination of these molecular events correlates viral capsid assembly with different conformations of the amphipathic alpha-helix in the precursor capsid, as a five-alpha-helix bundle at the pentamers or an open star-like conformation at the hexamers. These results, reminiscent of the assembly pathway of positive single-stranded RNA viruses, such as nodavirus and tetravirus, add new insights into the evolutionary relationships of dsRNA viruses.     

DNA sequence alterations responsible for the synthesis of thermosensitive VP1 in temperature-sensitive BC mutants of simian virus 40.

The segment of simian virus 40 (SV40) genome which is recognized as the BC domain encodes for the COOH-terminal end of the SV40 major capsid protein VP1. Mutations in this domain lead to the synthesis of a thermosensitive VP1 which fails to assemble mature SV40 at the nonpermissive temperature. We determined the DNA sequences of eight BC mutants and compared them with the DNA sequences of wild-type SV40, polyomavirus, and BK virus. We found that BC11 and BC223 mutations result from changes in nucleotide residues 2367 (A to C) and 2084 (C to T), respectively. The others (i.e., BC208, BC214, BC216, BC217, BC248, and BC274) share the same point mutation at nucleotide 2354 (C to T). These mutations resulted in the following changes: Lys to Thr, His to Tyr, and Pro to Ser at VP1 amino acid residues 290, 196, and 286, respectively.     

Characterization of tubule and monomer derived from VP4 protein of infectious bursal disease virus

The VP4 protein of infectious bursal disease virus (IBDV) is a serine protease that processes the polyprotein for viral assembly. VP4 has been found to associate primarily with type II IBDV tubules that are 24 nm in diameter. In this study, a chimeric VP4, assigned as HS1VP4, was constructed with a VP4-autocleavage site inserted between the N-terminal His-tag and the VP4 sequence. The results showed that the VP4 forms tubules after the self-cleavage of HS1VP4 when expressed in Escherichia coli. Furthermore, a deletion of 28 amino acids at the C-terminus of VP4 resulted in monomers and dimers instead of tubule formation; mutants of S652A and K692A at active site destroyed the activity. The endopeptidase activity of these monomers and dimers was approximately 12.5 times higher than that of VP4 tubules. Additionally, the formation of tubules inhibited VP4 protease activity, as demonstrated through in vitro assays. The production and characterization of monomers or dimers that have greater endopeptidase activity and protease activity than tubules can provide further insight into VP4 tubule assembly and the regulation of VP4 activity in host cells; this insight will facilitate the development of new anti-IBDV strategies.     

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

The capsid of infectious bursal disease virus (IBDV), a nonenveloped virus of the family Birnaviridae, has a T=13l icosahedral shell constituted by a single protein, VP2, and several disordered peptides, all derived from the precursor pVP2. In this study, we show that two of the peptides, pep11 and pep46, control virus assembly and cell entry. Deletion of pep11 or even simple substitution of most of its residues blocks the capsid morphogenesis. Removal of pep46 also prevents capsid assembly but leads to the formation of subviral particles formed by unprocessed VP2 species. Fitting with the VP2 atomic model into three-dimensional reconstructions of these particles demonstrates that the presence of uncleaved pep46 causes a steric hindrance at the vertices, blocking fivefold axis formation. Mutagenesis of the pVP2 maturation sites confirms that C terminus processing is necessary for VP2 to acquire the correct icosahedral architecture. All peptides present on virions are accessible to proteases or biochemical labeling. One of them, pep46, is shown to induce large structural rearrangements in liposomes and to destabilize target membranes, demonstrating its implication in cell entry.     

The oligomerization domain of VP3, the scaffolding protein of infectious bursal disease virus,plays a critical role in capsid assembly

Infectious bursal disease virus (IBDV) capsids are formed by a single protein layer containing three polypeptides, pVP2, VP2, and VP3. Here, we show that the VP3 protein synthesized in insect cells, either after expression of the complete polyprotein or from a VP3 gene construct, is proteolytically degraded, leading to the accumulation of product lacking the 13 C-terminal residues. This finding led to identification of the VP3 oligomerization domain within a 24-amino-acid stretch near the C-terminal end of the polypeptide, partially overlapping the VP1 binding domain. Inactivation of the VP3 oligomerization domain, by either proteolysis or deletion of the polyprotein gene, abolishes viruslike particle formation. Formation of VP3-VP1 complexes in cells infected with a dual recombinant baculovirus simultaneously expressing the polyprotein and VP1 prevented VP3 proteolysis and led to efficient virus-like particle formation in insect cells.