The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry
2009; Springer Nature; Volume: 28; Issue: 11 Linguagem: Inglês
10.1038/emboj.2009.109
ISSN1460-2075
AutoresS. Duquerroy, Bruno R. da Costa, Céline Henry, Armelle Vigouroux, Sonia Libersou, Jean Lepault, Jorge Navaza, Bernard Delmas, F.A. Rey,
Tópico(s)Animal Virus Infections Studies
ResumoArticle30 April 2009free access The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry Stéphane Duquerroy Stéphane Duquerroy Institut Pasteur, Unité de Virologie Structurale, Virology Department and CNRS URA 3015, Paris, France Université Paris-Sud, Faculté d'Orsay, Orsay Cedex, France Search for more papers by this author Bruno Da Costa Bruno Da Costa INRA UR892, Virologie et Immunologie Moléculaire, Jouy-en-Josas, France Search for more papers by this author Céline Henry Céline Henry INRA UR477, Unité BioBac, Jouy-en-Josas, France Search for more papers by this author Armelle Vigouroux Armelle Vigouroux Institut Pasteur, Unité de Virologie Structurale, Virology Department and CNRS URA 3015, Paris, FrancePresent address: CNRS UPR 3082 LEBS, 91190 Gif-sur-Yvette, France Search for more papers by this author Sonia Libersou Sonia Libersou CNRS-INRA UMR 2472, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, France Search for more papers by this author Jean Lepault Jean Lepault CNRS-INRA UMR 2472, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, France Search for more papers by this author Jorge Navaza Jorge Navaza CNRS-INRA UMR 2472, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, FrancePresent address: CNRS/CEA UMR 5075 IBS, 38027 Grenoble, France Search for more papers by this author Bernard Delmas Bernard Delmas INRA UR892, Virologie et Immunologie Moléculaire, Jouy-en-Josas, France Search for more papers by this author Félix A Rey Corresponding Author Félix A Rey Institut Pasteur, Unité de Virologie Structurale, Virology Department and CNRS URA 3015, Paris, France Search for more papers by this author Stéphane Duquerroy Stéphane Duquerroy Institut Pasteur, Unité de Virologie Structurale, Virology Department and CNRS URA 3015, Paris, France Université Paris-Sud, Faculté d'Orsay, Orsay Cedex, France Search for more papers by this author Bruno Da Costa Bruno Da Costa INRA UR892, Virologie et Immunologie Moléculaire, Jouy-en-Josas, France Search for more papers by this author Céline Henry Céline Henry INRA UR477, Unité BioBac, Jouy-en-Josas, France Search for more papers by this author Armelle Vigouroux Armelle Vigouroux Institut Pasteur, Unité de Virologie Structurale, Virology Department and CNRS URA 3015, Paris, FrancePresent address: CNRS UPR 3082 LEBS, 91190 Gif-sur-Yvette, France Search for more papers by this author Sonia Libersou Sonia Libersou CNRS-INRA UMR 2472, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, France Search for more papers by this author Jean Lepault Jean Lepault CNRS-INRA UMR 2472, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, France Search for more papers by this author Jorge Navaza Jorge Navaza CNRS-INRA UMR 2472, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, FrancePresent address: CNRS/CEA UMR 5075 IBS, 38027 Grenoble, France Search for more papers by this author Bernard Delmas Bernard Delmas INRA UR892, Virologie et Immunologie Moléculaire, Jouy-en-Josas, France Search for more papers by this author Félix A Rey Corresponding Author Félix A Rey Institut Pasteur, Unité de Virologie Structurale, Virology Department and CNRS URA 3015, Paris, France Search for more papers by this author Author Information Stéphane Duquerroy1,2, Bruno Da Costa3, Céline Henry4, Armelle Vigouroux1, Sonia Libersou5, Jean Lepault5, Jorge Navaza5, Bernard Delmas3 and Félix A Rey 1 1Institut Pasteur, Unité de Virologie Structurale, Virology Department and CNRS URA 3015, Paris, France 2Université Paris-Sud, Faculté d'Orsay, Orsay Cedex, France 3INRA UR892, Virologie et Immunologie Moléculaire, Jouy-en-Josas, France 4INRA UR477, Unité BioBac, Jouy-en-Josas, France 5CNRS-INRA UMR 2472, Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, France *Corresponding author. Unité de Virologie Structurale, Virology Department and CNRS URA 3015, 25 rue du Dr. Roux, Paris 75015, France. Tel.: +33 1 45688563; Fax: +33 1 45688993; E-mail: [email protected] The EMBO Journal (2009)28:1655-1665https://doi.org/10.1038/emboj.2009.109 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 Å X-ray structure of a picobirnavirus—an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two-fold symmetric dimers of a coat protein (CP) with a new 3D-fold. We show that, as many non-enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post-translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120-subunits capsid has evolved animal cell invasion properties. Introduction Double-stranded (ds) RNA viruses constitute a broad category of icosahedral viruses infecting a large spectrum of living organisms. The hosts range from prokaryotes—exemplified by the Pseudomonas phage ϕ6 in the Cystoviridae family of viruses—to humans, like rotaviruses (Reoviridae family (Estes and Kapikian, 2007), see http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm for virus taxonomy) or the more recently discovered picobirnaviruses (PBVs), which are associated with pathogenic symptoms like diarrhoea and gastroenteritis (Grohmann et al, 1993). Moreover, many of the primitive unicellular eukaryotes—yeast (McCabe et al, 1999) and protozoa (Wang and Wang, 1991)—contain cytoplasmic genetic elements that have been identified as simple capsid dsRNA viruses (as opposed to the complex capsid organization of viruses of the Reoviridae family (Estes and Kapikian, 2007; Roy, 2007; Schiff et al, 2007)). These simple-capsid dsRNA viruses never leave the cytoplasmic environment and are unable of invading a cell from outside. They are transmitted only during cell division or through cytoplasmic bridges that open during cell mating (McCabe et al, 1999). The best-studied example is L-A virus of yeast (Naitow et al, 2002), which belongs to the Totiviridae family. A characteristic feature of dsRNA virus particles in general is that their genome is always confined and transcribed within the particle, hidden from detection by the innate immunity sensors of the infected cell. On transcription, viral mRNAs are extruded through specific portals at the five-fold axes of the particle. This was experimentally shown for the yeast L-A virus (Fujimura et al, 1986) and also for the core particles of viruses in the Reoviridae family (Lawton et al, 2000) and the Cystoviridae family (Qiao et al, 2008). A number of elegant 3D structural studies have shown that the complex, multilayered capsid dsRNA viruses in the Reoviridae family (Grimes et al, 1998; Reinisch et al, 2000; Nakagawa et al, 2003; Yu et al, 2008) and also the ϕ6 bacteriophage (Huiskonen et al, 2006) share with the simple capsid totiviruses the quaternary organization of their inner capsid layer (Bamford et al, 2005), which has an icosahedral architecture made of 120 subunits. The two independent coat protein (CP) polypeptides in the icosahedral asymmetric unit of this core capsid layer make non-equivalent contacts with each other. This is in contrast to the packing symmetry observed in the majority of icosahedral virus particles analysed, which display a surface lattice triangulation originating from quasi-equivalent contacts between capsid proteins (Caspar and Klug, 1962). The 120-subunits icosahedral architecture is a hallmark of dsRNA viruses, and has not been observed in infectious virions of any other category of viruses. In the Reoviridae and Cystoviridae, this inner capsid is enclosed within a second icosahedral layer, which is organized with triangulation T=13. This second layer is in turn enclosed within a third layer in the case of rotaviruses. In this particular case, the third layer is used for membrane translocation when invading a new cell, delivering a double-layered 'core' particle, which becomes transcriptionally active in the cytoplasm of the target cell (Pesavento et al, 2006). Orthoreoviruses (the prototype members of the Reoviridae) use, instead, the capsid protein of the second layer (termed mu1) for entry, delivering a transcriptionally active single-layered core particle into the cytoplasm (Reinisch et al, 2000). Mu1 undergoes an activating autoproteolytic cleavage generating a myristoylated N-terminal peptide, which was shown to be critical for entry into cells (Odegard et al, 2004; Ivanovic et al, 2008). Notably different are the Birnaviruses (Coulibaly et al, 2005), which are dsRNA viruses having only a single-layered capsid of icosahedral surface symmetry of triangulation T=13. In this case, the capsid protein (CP) is also matured to release peptides from its C-terminal end that are used for cell invasion (Chevalier et al, 2005; Galloux et al, 2007). Birnaviruses are the exception among dsRNA viruses because they lack the inner 120-subunits layer, and the CP has a shell domain that is homologous to the CP of simple +sRNA insect viruses like the T=3 nodaviruses; yet its projecting domain is homologous to that of the counterpart in the T=13 second layer CP in the members of the Reoviridae. Here, we report the X-ray structure of a PBV particle, using recombinant virion-like particles (VLPs) obtained by expressing the rabbit PBV capsid protein gene in insect cells. PBVs are frequently detected in stool samples from children with diarrhoea (Bhattacharya et al, 2007; Finkbeiner et al, 2008; Giordano et al, 2008) as well as in samples from immunocompromised patients. Although PBVs are widespread in humans and mammals in general—as well as birds (Chandra, 1997; Masachessi et al, 2007)—our current knowledge of their biology is very limited, mainly because of their non-cultivable status, that is, the absence of a cell culture system for propagating the virus. The first complete nucleotide sequence of the two genomic segments of PBV isolated from humans was published in 2005 (Wakuda et al, 2005). Genomic segment 1 (which is between 2.3 and 2.6 kilobase-pairs long, depending on the genotype) has two open reading frames (ORFs). Although the first one codes for a protein of unknown function, the second one is shown here to encode the CP. The smaller segment 2 has a single-ORF coding for the viral polymerase. The crystal structure shows that the PBV particles have a single-layered icosahedral architecture made of 120 subunits, with the notable difference that the particles are formed by 60 two-fold symmetric dimers of a CP with a new fold—an organization not observed in any icosahedral virus crystallized so far. The architecture is clearly different to that of totiviruses and the inner layer of the Reoviridae, but appears related to that of the simple capsid dsRNA viruses of the Partitiviridae family (Ochoa et al, 2008), which infect yeast and plants. We show that a specific feature of PBV is that the CP undergoes an autocatalytic maturation releasing a positively charged, post-translationally modified peptide that remains associated within the virion, reminiscent of the process involved in the maturation of other non-enveloped animal viruses. Results PBV CP spontaneously assembles into VLPs The identity of the capsid protein coding gene in the PBVs had not been shown earlier. We cloned the second ORF present in segment 1 of rabbit PBV (Green et al, 1999) into a baculovirus vector for production in insect Sf9 cells. The protein encoded displays 25% amino-acid sequence identity with its human counterpart (see Figure 1A). CsCl gradient analysis of recombinant baculovirus infected cell extracts showed the presence of particles that segregate into two discrete bands corresponding to densities of 1.320 and 1.345 g/ml, termed low- (LD) and high-density (HD) bands, respectively. Electron microscopy observations of the material present in the HD band, which contained about 80% of the total material, showed homogeneous 35 nm diameter spherical particles. The diameter of the particles—as well as the overall morphology—is the same as those reported for intact PBV virions isolated from pigs (Ludert et al, 1991). This study thus experimentally identifies the second ORF of PBV genomic segment 1 as coding for the CP. Figure 1.Structure of the PBV CP. (A) Amino-acid sequence alignment of rabbit and human PBV CP (accession Q9Q1V2 and Q50LE5, respectively) highlighting conserved amino acids. A vertical arrow points to the N-terminal residue of the 55 kD CP found in the particle. Secondary structure elements (SSE) are labelled as α, η and β, for α-helices, 3/10-helices and β-strands, respectively, numbered sequentially from the N terminus and coloured to match the diagrams of the other panels. A labeled circle above the sequence marks residues approaching the icosahedral (I2, I3 and I5) and local (L2) symmetry axes. Small circles below the sequence flag residues involved in L2-related intra-dimer contacts (open circles) and between dimers in the capsid (full coloured circles: blue and red denote single and double (meaning two non-equivalent) contacts in the capsid, respectively. Red triangles below the sequence flag residues involved in the hydrogen-bonding network surrounding the transproteolytic cleavage site. (B) Ribbon diagram of the CP dimer. Shell and Projecting domains are labelled S and P, and the N- and C-termini are indicated. One subunit is coloured by SSE, with all helices in red except for the swapped region (η1-α1, orange) and β-sheets in yellow and green in the S domain, and purple, blue and pink in the P domain. In the blue sheet, dark blue indicates the swapped, N-terminal β1–β2 hairpin. For clarity, the second subunit is shown in grey. (C) Topology diagram. β-strands and α or η helices are indicated by arrows and cylinders, respectively, coloured and numbered as in A, and connected by a black line. A broken line connects elements that are swapped between the two subunits in the dimer. Pale red and blue ovals encompass secondary structure elements of the S and P domains, respectively. Download figure Download PowerPoint Particles in the HD band contained nucleic acid molecules of cellular origin ranging between 0.1 and 2 kb. In contrast, the material recovered from the LD band was a heterogeneous mix of different sizes and also broken particles containing essentially no nucleic acid. SDS–PAGE analysis revealed a protein of 55 kD present in both bands, as well as a small 7 kD protein present only in the HD band (Supplementary Figure S1). Mass spectrometry (MS) of the 7 kD polypeptide indicated that it consists of a post-translationally modified segment spanning the 65 N-terminal amino acids of CP (Supplementary Figure S2). N-terminal sequencing of the 55 kD protein showed that it corresponds to the remaining of the precursor molecule, amino acids 66–590, which will be referred to as the capsid protein (CP) in the rest of the manuscript. The presence of the 7-kD peptide was observed only when nucleic acid was packaged in the capsid, suggesting a role in genome packing—consistent with the numerous positively charged residues present in its amino-acid sequence, as can be seen in Figure 1A—in addition to a possible role in cell entry (see below 'Maturation of the CP'). Indeed, preliminary experiments showed that the purified PBV particles have a membrane disrupting activity, inducing leakage of liposome-encapsulated fluorescent dyes (Supplementary Figure S3). Moreover, as had been observed for rotaviruses (Nandi et al, 1992), trypsin treatment of the PBV particles augmented liposome leakage (Supplementary Figure S3). We were however unable to identify the trypsin cleavage site in the PBV particles. Structural studies VLPs recovered from the HD band were used for crystallization trials and for electron cryomicroscopy (cEM) analyses. Diffraction quality crystals were obtained as described in Materials and methods. An initial cEM reconstruction to 20 Å resolution (Figure 2B) was combined with X-ray diffraction data collected at synchrotron sources to extend phases using 30-fold averaging to a resolution of 3.4 Å. This procedure resulted in a very clear electron density map of the VLP (Supplementary Figure S4), allowing the trace of the CP polypeptide chain. No density corresponding to the 7-kD peptide was observed. The model was refined against structure factors measured between 50 and 3.4 Å resolution, the final statistics are shown in Table I. The final model contains two independent polypeptide chains, labelled A and B, which are not related by the icosahedral symmetry of the particle, with residues A66 to A590 and B66 to B586 (i.e. the last four residues of chain B are disordered). Figure 2.Surface features of the PBV capsid. (A) CP dimer with the subunits in different shades of grey. Top and bottom are views down the L2 axis, from outside and inside the VLP, respectively, with a side view in the middle. The internal curvature of the CP dimer matches roughly the internal radius of the particle. Note the intricate interface between subunits, generated by the N-terminal β1β2 and α1-η1 swapping, as indicated. The location of the N-terminus marks the site of the transproteolytic cleavage. (B) Surface of the VLP. cEM 3D-reconstruction showing the presence of 60 dimeric protrusions formed by domain P. The contour level was chosen to accommodate the volume of the CP dimer in the icosahedral asymmetric unit. A few symmetry axes are labelled for orientation. Top and bottom panels are seen down the I3 and I5 axes, respectively, slightly miss-oriented in order to help visualize certain features, like the cleft at the I2 axes, the I5 depression and the flat I3 region. Some of the symmetry axes are drawn, with standard symbols for 2-, 3- and 5-fod axes (empty ellipse, triangle and pentagon, respectively) and labelled. Bar: 100 Å. Download figure Download PowerPoint Table 1. Data collection and refinement statistics Data collection Space group P3221 Cell dimensions a, b, c (Å) 407.42, 407.42, 808.63 α, β, γ (deg) 90.0, 90.0, 120.0 Resolution (Å) 80.0–3.4 (3.6–3.4)a Rsym or Rmerge 24.0 (52.6)a I/σI 3.3 (1.0)a Completeness (%) 69.9 (41.6)a Redundancy 1.5 (1.3)a Refinement Resolution (Å) 50.0–3.4 No. of reflections 736678/3758 Rwork/Rfree b 27.2/27.3 No. atoms Protein 8234 Water 52 B-factors Protein 34.1 Water 5.0 RMS deviations Bond lengths (Å) 0.010 Bond angles (deg) 1.5 a Values in parentheses are for highest-resolution shell. b Rfree value calculated with 0.5% of the relflections data set, selected by resolution shells. Because of the high redundancy in the data set due to a 30-fold non-crystallographic symmetry, the Rfree does not represent a 'free' set, hence the very small difference with the working R. To avoid over-fitting, refinement was therefore carried out with a high weight to the geometry of the model, and the NCS were maintained as constraints throughout, to reduce the number of parameters to be fitted. The PBV capsid is built of 60 symmetric dimers The 3D structure revealed a particle made of 60 symmetric CP dimers displaying an intricate interface, with the N-terminal 55 residues (amino acids 66–120, see Figures 1 and 2A) exchanged between the two subunits. The trace of the polypeptide chain displays a complex topology (Figure 1B and C); a search in the protein database identified no entry with significant structural homology, indicating that the 3D-fold of PBV CP has not been observed earlier. The tertiary organization gives rise to a compact structure in which two domains can be recognized, a shell (S) domain that contains all of the α-helices and two β-sheets (yellow and green in Figure 1B). The remaining β-sheets are part of the projecting (P) domain, which forms the notable protrusions that stand out in the low resolution cEM 3D reconstruction (Figure 2B). The CP dimer is such that each domain is shared by the two subunits, with the N-terminal β-hairpin (β1–β2, dark blue in Figure 1) inserted in the P domain and the N-terminal helix-turn-helix motif (η1–α1, orange) in the S domain of the partner subunit in the dimer. This intricate interface, highlighted in Figure 2A, indicates that dimerization takes place during folding of the polypeptide chains, giving rise to an extensive buried surface of about 12400 Å2 (6200 Å2 per subunit), implying that the CP dimer is the building block for particle assembly. The inner surface of the dimer has a smooth curvature (Figure 2B, middle row) roughly matching that of the inner radius of the particle (130 Å, see also Figure 3). Although each subunit of the CP dimer makes non-equivalent contacts in the particle, there are only minor distortions of the local two-fold symmetry of the dimer, affecting mainly side-chains and loops in the contact regions (Supplementary Figure S5). The overall root mean square deviation after superposition of the refined models of the two subunits of the dimer is 0.95 Å for 521 α carbons; the rotation relating the two subunits is very close to 180.0 degrees (Supplementary Table S1). This axis is indicated by L2 (for 'local' two-fold axis, Figure 2B) to differentiate from the icosahedral symmetry axes, which are denoted by I2, I3 and I5 (for two-fold, three-fold and five-fold icosahedral axes, respectively) in the descriptions below. Figure 2B shows that the L2-related dimeric protrusions of CP are in turn related by the I2 axes, which lie in a deep cleft between adjacent CP dimers. The reconstruction also shows that there is a small depression at each of the I5 and a flat region at the I3 axes. Figure 3.Parallel dimers define the outline of a rhombus-shaped tile on the icosahedral particle. (A) Two CP dimers related by an I2 axis of the particle are displayed as ribbons, with the S and P domains coloured blue and red, respectively. The nearest icosahedral and local symmetry axes are drawn in projection and labelled. The left panel is projected down the I2 axis. The L2 axes make an angle of about 11.5 degrees with the I2 axis and do not intersect each other, but lie in parallel planes perpendicular to the paper; small arrows by the L2 labels indicate the direction and length (6.2 Å) of the translation needed to bring these planes into coincidence with a parallel vertical plane containing the I2 axis. The right panel shows an orthogonal view rotated about a vertical axis. Full-line arcs indicate the inner and outer radii of the particle (labelled), highlighting the curvature of the diamond tile. The L2 axes cross the vertical plane containing the I2 axis (which is in the plane of the paper) along a vertical line, at a distance of 14.5 Å from each other, above and below the I2 axis. This vertical line intersects the I2 axis at a radius of about 35 Å (broken line arc), indicating a sharper local curvature at the I2 contact than that of the overall particle. The particle centre is indicated by O, where I5, I3 and I2 axes intersect. Residues Asn 157 and Asn 221 are displayed as yellow and red spheres and labelled, as well as helices α5 and α6 mentioned in the text. C-ter labels the last four amino acids of chain A (587–590, disordered in chain B) running along the I5 axis. Bar: 100 Å. (B). Triacontahedral design of the PBV particle, each of the 30 rhombic tiles is coloured differently. The red tile is oriented as the one displayed in the top panel. (C) Convergence with the icosahedral organization with the flavivirus particle, where each of the 30 tiles (coloured as in B) is composed of 3 parallel dimers of the envelope protein. Coordinates obtained from PDB entry 1THD. Panels B and C are at the same scale. Bar: 500 Å. Download figure Download PowerPoint Conserved segments are involved in capsid contacts The alignment of the two available sequences of PBV CP (rabbit and human) displayed in Figure 1A shows blocks of amino-acid conservation spread apparently at random throughout the primary structure of the protein. In contrast, in the 3D structure these patches display a consistent pattern, mapping to regions that are mainly involved in dimer–dimer contacts in the icosahedral capsid (indicated with small full circles underneath the sequence), whereas the intra-dimer contacts (small open circles) appear less conserved. One exception is the conserved block at the centre of helix α10, buried within the CP subunit, which includes residues participating in the hydrogen-bonding network surrounding the transproteolytic cleavage site used to generate the CP N terminus (see below and Supplementary Figure S6). The conservation pattern also shows that the P domain spans the least conserved regions of the polypeptide chain, consistent with being exposed and likely to undergo antigenic drift, as well as being potentially used for receptor binding with different specificities in viruses infecting different hosts. Important conserved motifs are located in regions approaching the icosahedral axes of the particle. For instance, the α2–α3 loop has the conserved sequence 157-NSG-159 lying right at the I5 axis (Figures 1A and 3A). The structure shows that Gly 159 is conserved because side chain atoms would collide with their five-fold-related counterparts. The side chain of the preceding Ser 158 contributes to a ring of interactions about the I5 axis, whereas its main chain carbonyl hydrogen bonds the main chain amide of Gly 159 of the neighbouring subunit, in a β interaction that results in a thin five-fold β-annulus sealing the I5 depression of the particle. Finally, the Asn 157 side chain hydrogen bonds its main chain carbonyl, stabilizing the polypeptide chain in the conformation required to allow the described interactions formed by the immediately downstream residues. The five α2–α3 loops appear to constitute a sort of diaphragm—as seen down the I5 axis (Figure 4). The I5 depression leaves room for a concerted movement of the five loops to open the diaphragm by breaking open the five-fold hydrogen bonds of the β-annulus, triggered by a putative conformational change during transcription, as is the case in other dsRNA viruses (see Discussion). Figure 4.View of the PBV particle down an I5 axis. The individual subunits are coloured according to secondary structure elements: α-helices blue, β-sheets red and random coil orange/light brown. Note the I5 loop with the conserved 157-NSG-159 segment at the centre, forming a diaphragm-like structure at the I5 axis. Bar: 100 Å Download figure Download PowerPoint At the I3 axes, the side chain of conserved Asn 221 breaks the short helix α5 by capping its C-terminal end, resulting in a 120 degrees kink of the polypeptide chain, stabilizing this corner of the CP dimer and contributing to the compact packing of dimers at the particle surface (Figure 3). The conserved residues at both I5 and I3 axes display a nearly identical conformation at the L2-related site (see Supplementary Figure S3). Moreover, the conserved Asn 159 and Asn 221 (at the I5 and I3 axes, respectively) come together to contact each other at the L2-related site (highlighted in Figure 3A). In summary, considering the limited sequence identity between human and rabbit PBVs (25%), the conservation pattern helps in pointing to residues with likely functional properties. Maturation of the CP Many non-enveloped animal viruses undergo an autoproteolytic cleavage reaction, which activates the virion for entry usually by priming the particle for the release of a membrane-active peptide on interactions with the host. This maturation step often takes place on assembly of the particle. The PBV CP structure provides evidence for intra-dimer, transproteolytic cleavage between residues 65 and 66 (arrow in Figure 1A). The N-terminal residue Asp 66 of one subunit remains in the presumed catalytic site of the opposite subunit in the dimer. The side chain of Asp 66 points to solvent at the interior of the particle. The very clear experimental electron density map shows that the charged amino-terminal group of the polypeptide chain is positioned to make a bidentate hydrogen bonding interaction (and salt bridge) with a conserved buried glutamic acid, Glu 439 of the partner subunit (Supplementary Figure S6). This interaction stabilizes the post-cleavage structure. Proteolysis takes place C-terminal to conserved Asn 65 (Figure 1A), similar to the described autocatalytic nodavirus and tetravirus endopeptidase activity (Reddy et al, 2003), in which cleavage occurs after a conserved asparagine residue (Hosur et al, 1987; Agrawal and Johnson, 1992). The proposed catalytic mechanism involves a carboxylate group (the side chain of an aspartic or glutamic acid) of CP that gets positioned during assembly such that
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