Birnavirus VP4 Processing Endopeptidase
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ABSTRACT:
SUBMITTER: Delmas B
PROVIDER: S-EPMC7150322 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Viruses of the Birnaviridae family are characterized by their bisegmented double-stranded RNA genome that resides within a single-shelled non-enveloped icosahedral particle. They infect birds, aquatic organisms, and insects. Tellina virus 1 (TV-1) is an Aquabirnavirus isolated from the mollusk Tellina tenuis. It encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is cleaved by the self-encoded protease VP4 to yield capsid precursor protein pVP2, peptide X, and ribonucleoprotein VP3. Here we report the crystal structure of an intramolecular (cis) acyl-enzyme complex of TV-1 VP4 at 2.1-? resolution. The structure reveals how the enzyme can recognize its own carboxyl terminus during the VP4/VP3 cleavage event. The methyl side chains of Ala830(P1) and Ala828(P3) at the VP4/VP3 junction point into complementary shallow and hydrophobic S1 and S3 binding pockets adjacent to the VP4 catalytic residues: nucleophile Ser738 and general base Lys777. The electron density clearly shows that the carbonyl carbon of Ala830 is covalently attached via an ester bond to the O? of Ser738. A highly ordered water molecule in the active site is coordinated in the proper position to act as the deacylating water. A comparative analysis of this intramolecular (cis) acyl-enzyme structure with the previously solved intermolecular (trans) acyl-enzyme structure of infectious pancreatic necrosis virus VP4 explains the narrower specificity observed in the cleavage sites of TV-1 VP4.
| S-EPMC3069450 | biostudies-literature
Project description:Birnaviruses are unconventional members of the icosahedral double-stranded (dsRNA) RNA virus group. The main differential birnavirus trait is the lack of the inner icosahedral transcriptional core, a ubiquitous structure conserved in all other icosahedral dsRNA viruses, that shelters the genome from cellular dsRNA sensors and provide the enzymatic machinery to produce and extrude mature messenger RNAs. In contrast, birnaviral particles enclose ribonucleoprotein (RNP) complexes formed by the genome segments, the dsRNA-binding VP3 polypeptide and the virus-encoded RNA polymerase (RdRp). The presence of RNPs suggests that the birnavirus replication program might exhibit significant differences with respect to those of prototypal dsRNA viruses. However, experimental evidences supporting this hypothesis are as yet scarce. Of particular relevance for the understanding of birnavirus replication is to determine whether RNPs act as intracellular capsid-independent transcriptional units. Our study was focused to answer this question using the infectious bursal disease virus (IBDV), the best characterized birnavirus, as model virus. Here, we describe the intracellular assembly of functional IBDV RNPs in the absence of the virus-encoded VP2 capsid polypeptide. Recombinant RNPs are generated upon coexpression of the IBDV VP1 and RdRp polypeptides and transfection of purified virus dsRNA. Presented data show that recombinant RNPs direct the expression of the IBDV polypeptide repertoire and the production of infectious virus in culture cells. Results described in this report constitute the first direct experimental evidence showing that birnaviral RNPs are intracellularly active in the absence of the virus capsid. This finding is consistent with presented data indicating that RNP formation precedes virus assembly in IBDV-infected cells, and supports the recently proposed IBDV replication model entailing the release of RNPs during the initial stages of the infection. Indeed, results presented here also support the previously proposed evolutionary connection between birnaviruses and positive-strand single-stranded RNA viruses.
| S-EPMC3907549 | biostudies-other
Project description:Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.
| S-EPMC1135784 | biostudies-other
Project description:Inhibitor studies were performed on the two endopeptidase activities involved in proinsulin conversion in isolated insulin secretory granules [Davidson, Rhodes & Hutton (1988) Nature (London) 333, 93-96]. The active-site-directed peptides L-alanyl-L-arginyl-L-arginylmethyldimethylsulphonium and L-alanyl-L-lysyl-L-arginylmethyldimethylsulphonium inhibited these activities in accordance with the observed cleavage pattern, suggesting that the primary amino acid sequence of the dibasic site was an important determinant of the endopeptidase substrate specificities.
| S-EPMC1138356 | biostudies-other
Project description:The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.
| S-EPMC7552023 | biostudies-literature
Project description: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.
| S-EPMC4457844 | biostudies-literature
Project description:Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5(*) and VP8(*) in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8(*) portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5(*), at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.
| S-EPMC114321 | biostudies-literature
Project description:In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall ('autolysins') are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall--acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate--sphere formation vs. lysis--after treatment with antibiotics that target cell wall synthesis.
| S-EPMC4401780 | biostudies-literature
Project description:Espirito Santo virus (ESV) is a newly discovered virus recovered as contamination in a sample of a virulent strain of dengue-2 virus (strain 44/2), which was recovered from a patient in the state of Espirito Santo, Brazil, and amplified in insect cells. ESV was found to be dependent upon coinfection with a virulent strain of dengue-2 virus and to replicate in C6/36 insect cells but not in mammalian Vero cells. A sequence of the genome has been produced by de novo assembly and was not found to match to any known viral sequence. An incomplete match to the nucleotide sequence of the RNA-dependent RNA polymerase from Drosophila X virus (DXV), another birnavirus, could be detected. Mass spectrometry analysis of ESV proteins found no matches in the protein data banks. However, peptides recovered by mass spectrometry corresponded to the de novo-assembled sequence by BLAST analysis. The composition and three-dimensional structure of ESV are presented, and its sequence is compared to those of other members of the birnavirus family. Although the virus was found to belong to the family Birnaviridae, biochemical and sequence information for ESV differed from that of DXV, the representative species of the genus Entomobirnavirus. Thus, significant differences underscore the uniqueness of this infectious agent, and its relationship to the coinfecting virus is discussed.
| S-EPMC3302246 | biostudies-literature
Project description:Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1*03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML.
| S-EPMC7078166 | biostudies-literature