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How baculovirus polyhedra fit square pegs into round holes to robustly package viruses

2009; Springer Nature; Volume: 29; Issue: 2 Linguagem: Inglês

10.1038/emboj.2009.352

1460-2075

Xiaoyun Ji, Geoff Sutton, Gwyndaf Evans, Danny Axford, Robin L. Owen, David I. Stuart,

Entomopathogenic Microorganisms in Pest Control

Article3 December 2009free access How baculovirus polyhedra fit square pegs into round holes to robustly package viruses Xiaoyun Ji Xiaoyun Ji Division of Structural Biology, University of Oxford, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford, UK Search for more papers by this author Geoff Sutton Geoff Sutton Division of Structural Biology, University of Oxford, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford, UK Search for more papers by this author Gwyndaf Evans Gwyndaf Evans Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author Danny Axford Danny Axford Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author Robin Owen Robin Owen Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author David I Stuart Corresponding Author David I Stuart Division of Structural Biology, University of Oxford, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford, UK Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author Xiaoyun Ji Xiaoyun Ji Division of Structural Biology, University of Oxford, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford, UK Search for more papers by this author Geoff Sutton Geoff Sutton Division of Structural Biology, University of Oxford, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford, UK Search for more papers by this author Gwyndaf Evans Gwyndaf Evans Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author Danny Axford Danny Axford Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author Robin Owen Robin Owen Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author David I Stuart Corresponding Author David I Stuart Division of Structural Biology, University of Oxford, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford, UK Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK Search for more papers by this author Author Information Xiaoyun Ji1, Geoff Sutton1, Gwyndaf Evans2, Danny Axford2, Robin Owen2 and David I Stuart 1,2 1Division of Structural Biology, University of Oxford, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford, UK 2Diamond Light Source Ltd, Diamond House, Harwell Science and Innovation Campus, Didcot, UK *Corresponding author. Division of Structural Biology, University of Oxford, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford OX3 7BN, UK. Tel.: +44 1865 278 567; Fax: +44 1865 278 547; E-mail: [email protected] The EMBO Journal (2010)29:505-514https://doi.org/10.1038/emboj.2009.352 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles. Introduction The virions of a number of insect viruses from disparate virus families (including baculoviruses, cytoplasmic polyhedrosis viruses (CPVs) and entomopoxviruses) are specifically occluded within robust crystalline particles (Rohrmann, 1986). Typically 1–3 μm in size, these tend to have a characteristic shape, and the well-ordered lattices are built mainly from a single viral protein (Di et al, 1991; Anduleit et al, 2005). The occlusion bodies act as protective packages allowing infectious virions to survive for long periods in harsh environments, providing a delivery system between hosts by oral-faecal routes and resisting solubilization until exposed to the alkaline insect midgut (Rohrmann, 1986). Baculoviruses are a family of large DNA viruses, which replicate and assemble within the host nucleus. They are divided into two genera, alpha- and beta-baculoviruses, referred to as alphaBV and betaBV, respectively (Jehle et al, 2006). During an infection, the cylinder-shaped (30–50 nm diameter, 200–300 nm long) dsDNA-containing nucleocapsids can be embedded while within the nucleus of the insect cell, in occlusion bodies, named polyhedra for alphaBVs (Rohrmann, 2008). AlphaBV polyhedra are usually 0.15–3 μm in size (Ackermann and Smirnoff, 1983) and contain virus nucleocapsids surrounded by an envelope derived from the nuclear membrane into which are incorporated at least five different virus-encoded proteins, some of which presumably interact with polyhedrin (the polyhedra protein) to orchestrate occlusion. Some baculovirus strains pack a single virion within this envelope, whereas others (the so-called multiple nuclear polyhedrosis viruses) pack a bunch of ∼5–15. Overall, the total number of virions in a polyhedra ranges from one to several hundred (Ackermann and Smirnoff, 1983), scattered apparently randomly within the polyhedra (Scharnhorst et al, 1977). Finally, the entire alphaBV polyhedra is wrapped in a sheath of carbohydrate and virus-encoded proteins (Gross et al, 1994). The ∼29 kDa polyhedrin is one of the most conserved proteins of the virus (Supplementary Figure 1). Naturally occurring amino-acid substitutions in polyhedrin, many of them are single point mutations, produce a variety of phenotypic changes ranging from large, cuboid polyhedra, which occlude no or few virions (Carstens et al, 1986; Katsuma et al, 1999; Lin et al, 2000), to overall changes in polyhedra shape (Cheng et al, 1998). A nuclear localization signal (NLS), K32RKK35, directs the polyhedrin to the nucleus (Jarvis et al, 1991). The structure of the polyhedrin and its organization within the polyhedra has remained obscure, although analysis of partially dissolved baculovirus polyhedra suggested the presence of dodecameric or disulphide-linked octameric molecules of polyhedrin (Rohrmann, 1977; Scharnhorst and Weaver, 1980). In comparison with the rod-shaped baculoviruses, CPV is 75 nm in diameter and the dsRNA genome is contained in an icosahedral protein capsid, which is occluded directly into the non-enveloped polyhedra (Mertens et al, 2005). Similar to baculoviruses, the shape of CPV polyhedra can be altered by point mutations of polyhedrin (Ikeda et al, 1998). The crystal structures of both virus-containing and recombinant empty Bombyx mori CPV type 1 polyhedra have been determined (Coulibaly et al, 2007), revealing the polyhedra to be beautifully assembled from rigid 28.6 kDa polyhedrin building blocks, which assemble with the aid of ribonucleoside triphosphates (NTPs) to form a lattice that contains virtually no solvent. The polyhedrin is formed from a β-barrel core with helical extensions and was dissimilar to any other known protein structure. The polyhedrin lattice possesses cubic symmetry, so that its strength will be maximally isotropic and it matches, as closely as is possible in a crystal lattice, the icosahedral symmetry of the CPV particle. Remarkably, X-ray powder diffraction analysis of baculovirus and CPV polyhedra (Fujiwara et al, 1984; Di et al, 1991; Anduleit et al, 2005) indicated that, despite no obvious amino-acid sequence similarity, they possess crystal lattices, which are essentially indistinguishable in terms of size (a=10 nm) and symmetry (body centred lattice with 23 symmetry), suggesting that these parameters, which presumably underpin the biological properties of the polyhedra, are strongly conserved. To establish whether this arises, as has been predicted (Anduleit et al, 2005; Coulibaly et al, 2007), from similarities in the 3D structures of the proteins that build the lattice, we have determined such structures for wild-type and mutant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) polyhedra and find, surprisingly, that while the space group is identical to CPV polyhedrin (I23), AcMNPV polyhedrin in fact resembles more closely the canonical capsid protein structure of the picornavirus lineage of viruses (Bamford et al, 2005). Although not providing conclusive evidence for the evolutionary relationships between these viral polyhedra, the AcMNPV polyhedrin structure explains various functional aspects, including pH-dependent disassembly and adaptation to the nuclear life style of baculoviruses. Results Structure determination We have determined, from in vivo grown crystals, the structures of both virus-containing wild-type and a virus-empty mutant (G25D) polyhedra from AcMNPV, the type species of alphaBVs. The crystals were derived from infected insect cells (see Materials and methods), with the wild-type crystals being usually <5 μm in maximum linear dimension while many G25D crystals attained 5–10 μm. Despite their still tiny size, it was possible to determine the high-resolution (1.84 Å) structure for the larger mutant polyhedra by X-ray analysis using a tuneable micro-focus synchrotron beam (we used conventional seleno-methionine (Se-Met) labelling to solve the phase problem), Table 1. Owing to the small size of the crystals and their low-solvent content (nominally 21%), structure determination was challenging; however, the final structure is reliable (Rwork=0.164, Rfree=0.217 from 36.3 to 1.84 Å resolution, Table 1). Although the majority of the structure is precisely determined, the electron density is poorly defined for residues 3–7 (for which only the backbone could be traced) and 142–147, whereas residues 1–2, 32–48 and 174–186 could not be positioned at all. The final 1.8 Å resolution structure contains 88 water molecules, with no ions, buffer molecules or other small molecules visible. The structure of wild-type AcMNPV polyhedra was then determined at 3.0 Å resolution by molecular replacement (Table 1). The structures of mutant and wild-type polyhedra are very similar and because of its higher quality, we usually describe the mutant structure below; however, the wild-type structure is a little better packed (as described below) such that some of the disordered residues are clearly visible and this structure helped in the final interpretation of these regions. Table 1. Data collection, phasing and refinement statistics G25D nativea G25D Se-Meta Wild-type nativea Data collection Space group I23 Cell dimensions a=b=c (Å) 102.58 102.60 101.56 Resolution (Å) 50.0–1.84 (1.91–1.84) 40.0–3.0 (3.11–3.00) 50.0–3.0 (3.11–3.00) Unique reflections 15809 (1576) 3721 (366) 3329 (326) Rmerge 0.210 (0.000b) 0.248 (0.629) 0.383 (0.000b) I/σI 12.1 (1.0) 23.4 (7.9) 4.4 (1.4) Completeness (%) 100 (100) 100 (100) 99.6 (100) Redundancy 14.7 (10.9) 24.3 (24.3) 6.8 (6.4) Refinement Resolution (Å) 36.3–1.84 41.5–3.0 Number of reflections 15762 3325 Rwork/Rfree 0.164/0.217 0.205/0.238 Number of atoms Protein 1752 1723 Water 88 B-factors Protein 34.1 28.8 Water 34.5 r.m.s. deviations Bond lengths (Å) 0.007 0.007 Bond angles (deg) 1.04 0.94 Values in parentheses are for the highest resolution shell. a Seventeen G25D native crystals, 31 G25D Se-Met crystals and seven wild-type native crystals were merged, respectively. b R⩾1. Subunit structure AcMNPV polyhedra belong to space group I23 with one polyhedrin subunit in the crystal asymmetric unit, so that 24 copies of the 28.6 kDa (245 amino acids) subunit are tightly packed in the unit cell. The polyhedrin subunit can be broken down into three parts: N-terminal head, a β-barrel body (comprising the majority of the subunit) and C-terminal tail (Figure 1A and B). The β-barrel body is reminiscent of that of the capsid proteins of picorna-like viruses and we retain that strand nomenclature here. Four β-strands (BIDG) form the core of an extended first sheet with strands CHEF forming the second sheet, which contains fewer, shorter strands (Supplementary Figure 2A). Three helices (α2, α3 and η2) cap the exposed side of the sheets, enclosing the hydrophobic core and forming a groove between α2 and the BIDG sheet. Extending from strand βI is a C-terminal hook comprising 13 highly conserved residues (residues 233–245, Figure 1F; Supplementary Figure 1). The head domain (residues 1–31 and 49–63) consists of a hook-shaped loop, three anti-parallel β-strands (A, A′, A″), an α-helix and a 310 helix. As residues 32–48 were not seen in the electron density map, there is an ambiguity in connecting the N-terminal residues 1–31 to the rest of the subunit. In principle, 12 different links are feasible; however, for clarity, only one of these is shown in Figure 1A and B (we justify this choice below). Figure 1.Overview of structure. (A, B) The structure of AcMNPV polyhedrin, in two orthogonal views, shown as cartoon representations coloured from blue at the N-terminus to red at the C-terminus. Residues 3–7, which were built as poly-ala are drawn in grey. Disordered segments D1 (residues 32–48) and D2 (residues 174–186) are shown as dots. (C, D) Cartoon and surface representations of polyhedrin trimers shown in two orientations aligned to (E). Subunits are coloured by chain with one in each trimer coloured as in (A). (E) Dodecameric unit of four trimers linked by disulphide bonds (represented as orange spheres). The unit cell and three-fold axes are drawn to facilitate observation. (F) Amino-acid sequence conservation between the type species of the alphaBVs (AcMNPV) and betaBVs (CpGV). Conserved residues drawn in red boxes, similar residues in red type. The secondary structure assignment for AcMNPV is drawn above. Conserved cysteine is highlighted in yellow. Interactions involved in various stages of oligomization are mapped as lines coloured thus; purple for trimer, green for dodecamer, red for interactions through C-terminal and yellow for other interactions in the unit cell. The position of the nuclear localization signal (NLS) is indicated and residues exposed to the cavity are marked by blue triangles. The sequences were aligned with ClustalW (Chenna et al, 2003) and visualized using ESPript (Gouet et al, 1999). Download figure Download PowerPoint Mapping functional mutations A number of naturally occurring mutations have been described for baculovirus polyhedrin, some of the best characterized are described in Table 2. A significant number fall into one of the two classes: (i) mutations, which disrupt the formation of polyhedra and (ii) mutations, which result in the formation of larger polyhedra, sometimes in the cytoplasm. In general, mutations of class (i) tend to occur within the core β-barrel of the polyhedrin, in which they presumably significantly disrupt the molecule (Supplementary Figure 3); an example is the valine to phenylalanine mutation at residue 118 in which it would be difficult to accommodate the bulk of the aromatic side chain. In contrast, mutations of class (ii) tend to cluster in or near the disordered regions of the structure (Supplementary Figure 3). We have obtained structures for the wild-type virus and for one class (ii) mutant, G25D. The mutant polyhedra are larger than the wild-type and are found, empty of viruses, in the nucleus of the infected cells. Structurally, the change from glycine to aspartic acid is quite modest, and yet there are significant differences between the two types of polyhedra—in the wild–type, the smaller glycine amino acid provides space for a nearby side chain (Asp51), resulting in a better local packing and a slight (∼1 Å) reduction in the size of the unit cell. Although this change is small, it is completely reproducible and the changes observed are likely to be representative of the situation in vivo. Table 2. List of AcMNPV and BmNPV polyhedra mutant viruses adapted from Ribeiro et al (2009) Polyhedrin mutation Position in structure Virus occlusion Occlusion morphology Reference Class i M29/AcMNPV L85P Within βC (core) No Dispersed protein mass Carstens (1987) vSynlitx1B12P/AcMNPV V118F Within βD (core) No Dispersed protein mass Ribeiro et al (2009) #136/BmNPV L141F Within βE (core) No Dispersed protein mass Katsuma et al (1999) Class ii AcMNPV-Tkmt513/AcMNPV G25D Within α1, close to D1 No Large and cuboid Lin et al (2000) M5/AcMNPV P59L Within loop between βA″ and B Yes/no Large and cuboid Carstens et al (1986) #211/BmNPV E144L Within loop between βE and E′ Yes Irregularly shaped or small polyhedra Katsuma et al (1999) #128/BmNPV L171P Close to D2 No As wild-type occlusion Katsuma et al (1999) Others #220/BmNPV D58N I222T Within loop between βA″ and B Within βI Yes Cuboid Katsuma et al (1999) #126/BmNPV C178Y Within D2 No Dispersed protein mass Katsuma et al (1999) M934/AcMNPV L183F Within D2 No Dispersed protein mass Carstens et al (1992) A structural similarity to viral coat proteins, and perhaps to CPV polyhedrin Automated structural comparisons against the Protein Data Bank (Dali, Holm et al, 2008; SSM, Krissinel and Henrick, 2004) reveal that the AcMNPV polyhedrin has a jelly-roll fold similar to that observed in the capsid proteins of picorna-like viruses (Figure 2A; Supplementary Table I). Indeed, the most similar structures tend to be picornavirus VP3 major coat proteins and the 'insect picornavirus' cricket paralysis virus coat proteins. More detailed comparison with the program SHP (Stuart et al, 1979) reveals that the closest similarity is with VP2 of cricket paralysis virus, 119 equivalent Cαs can be superposed with r.m.s. deviation 3.2 Å. Although there are many structural differences, there are also points of similarity beyond the basic sheet topology, thus the BIDG sheet is the more extended in both the picornavirus capsid proteins and AcMNPV polyhedrin, and the topological positions of some of the helices match. This similarity is of a level that has earlier been used to infer divergence from a common ancestor, and this is graphically shown in Figure 3. A somewhat lower level of similarity is shown with CPV polyhedrin (Figure 2B), 86 Cαs superpose with r.m.s. deviation 3.5 Å. The Dali Z-score for this comparison is correspondingly low at 1.5 (Supplementary Table I); however, the program does not find any structures with significantly greater similarity to CPV polyhedrin and detailed SHP analysis confirms that cellular jelly-roll proteins such as tumour necrosis factor (TNF) are no more similar (Figure 3). Figure 2.Structural superpositions. Stereo views showing the alignment of (A) AcMNPV polyhedrin (orange) with cricket paralysis virus VP2 (green) (Tate et al, 1999) and (B) AcMNPV polyhedrin (orange) with CPV1 polyhedrin (purple). The program SHP (Stuart et al, 1979) was used to perform the superpositions. (C) Schematic representation of the packing of both AcMNPV and CPV1 polyhedrin trimers (shown as blocks) within their unit cells. The trimeric units are positioned rather differently on the three-fold body diagonal—this is represented in the central panel in which the centres of the trimers of differing polarity are reflected into the (0,0,0) to (½, ½, ½) portion. The AcMNPV trimer centres (shown in shades of green) lie close to (¼, ¼, ¼), whereas the CPV1 trimers (centres shown in shades of blue) are offset by about 6 Å. This accounts for the differences seen in the outer panels. Download figure Download PowerPoint Figure 3.Phylogenetic tree showing the relationship between the polyhedrin proteins of AcMNPV and CPV1, against picorna-like virus capsid proteins. Superpositions were performed using SHP (Stuart et al, 1979) and the phylogenetic tree calculated with PHYLIP (Felsenstein, 1989). The following abbreviations are used: AcMNPV, Autographa californica baculovirus; BEV, bovine enterovirus; ch, chain; COV, coxsackievirus A3; CPV1, cytoplasmic polyhedrosis virus type 1; CrPV, cricket paralysis virus; Echo, echovirus; FMDV, foot-and-mouth disease virus; Mengo, mengo virus; Polio, poliovirus; Rhino, rhinovirus; SBMV, southern bean mosaic virus; SV, swine vesicular disease virus; TME, theiler murine encephalomyelitis virus; TNF, tumour necrosis factor. Note that CPV1 polyhedrin is closest to AcMNPV polyhedrin, which is, however, closer to the picornavirus capsid structures than CPV1. Download figure Download PowerPoint Subunit organization in the crystal lattice The AcMNPV subunits form tightly packed trimers, which resemble a cube with one quadrant removed—this region houses the disordered portions of the molecule, as we discuss below (Figures 1C, D and 4). The jelly-roll β-barrels of each subunit are packed orthogonal to each other (reminiscent to that seen in the non-viral TNF trimer; Jones et al, 1989), and define the basic size of the crystal building block. The extent of the interface in the trimer depends on the assignment of the linkage of the N-terminal head, which as mentioned above is ambiguous, as 17 residues are missing and these occupy a region close to the crystallographic origin in which four three-fold and three two-fold symmetry axes converge, so that 12 different links are in principle feasible (it is conceivable, but unlikely that within a crystal the N-terminal residues are not always connected in the same way). Nevertheless, given the positioning of the symmetry axes, the distances involved and the nature of the subunit interactions described below, we consider it most likely that the N-terminal portion is attached to one of the subunits within a trimer (Supplementary Figure 4). The N-terminal head contributes to the clamping sheet fundamental to the trimer assembly, part of a robust trimer interface comprising a mixture of hydrophobic interactions, salt bridges and hydrogen bonds, which buries between 20 and 30% (∼3000–4000 Å2) of the surface area of each polyhedrin subunit (Figure 1F; Supplementary Figure 5). At the base of the trimer, the N-termini come together to form a short stalk. Helices α1 and η1 form the base and strands βA, A′ and A″ the upper region (these N-terminal strands clamp onto and expand the BIDG sheet from adjacent subunits; Supplementary Figure 2B). The C-terminal hooks protrude at right angles from three edges of the trimer cube. Figure 4.Schematic representations of polyhedra organization. Polyhedrin trimers are depicted as simplified cubic blocks, with the C-terminal hooks and pockets present. To clarify interpretation, the edges of the unit cell are shown in gold and a cyan tetrahedron symbolizes the cell centre. Within a unit cell, disulphide-linked trimers with one polarity are coloured light beech (A) and those with the opposite polarity are coloured light brown (C). (B) All eight trimers in the unit cell. The disulphide bond connecting adjoining trimers is shown as a dowel. (D) The crystal lattice is built up from repeats of the dodecameric unit. (E) Sketch of a cross-section through a polyhedron. The lattice spacing of the unit cells is illustrated as a dot pattern into which are embedded nucleocapsids (dark blue) surrounded by an envelope (cyan). (F) Light microscopy image of G25D mutant AcMNPV polyhedra. Download figure Download PowerPoint Eight of the cube-like trimers are arranged neatly to fill the crystal unit cell (Figure 4). These eight trimers in fact form two nested sets of four, one arrayed at the corners of the cell and the other at the centre, the only difference between these two sets being in the polarity of the trimer with respect to the three-fold axes (four trimers point inwards towards the centre of the unit cell, whereas the other four point outwards towards the corners). The regularity of the packing seen in Figure 4 arises because the trimers are situated almost exactly mid-way along the body diagonal between the centre and the corner of the crystal cell (Figures 1E and 2C). This arrangement leaves the C-terminal hooks jutting outwards and brings the absent quadrants of the trimers together around the centre and corner points, to form cavities (volume 110 000 Å3), which harbour the disordered portions of polyhedrin (Figure 4C; Supplementary Figure 6). Assuming that the N-terminal heads exchange within trimers, the closest interactions between trimers of the same polarity come at the centre of each face of the unit cell in which the βD-βE and βH-βI loops of subunits from neighbouring trimers come together. The interaction here is not extensive (800 Å2) and would not normally be sufficient to achieve a strong interaction; however, four salt bridges between highly conserved glutamate, arginine and asparagine side chains and a covalent disulphide lock between two symmetry-related copies of Cys132 tether this extended dimer (Figure 5A). Crystallographic and biochemical analyses show that in wild-type polyhedra, the disulphide is fully formed, whereas in the mutant polyhedra, it is partly reduced (Figure 5B). As Cys132 is the only cysteine fully conserved across both the alpha- and beta-baculoviruses (Supplementary Figure 1), it seems likely that this disulphide stabilization is biologically relevant. These interactions link four trimers of the same polarity in a tetrahedral arrangement to form a dodecameric cage (Figures 1E and 4A). Dodecamers, and smaller oligomers, can be observed by non-reducing SDS–PAGE with shorter heating times (Figure 5B) and chemical cross-linking (Scharnhorst and Weaver, 1980). The formation of the disulphide bonds between trimers presumably occurs relatively late, after disruption of the reducing environment of the nucleus. The assembly of the crystal is completed by clipping dodecamers together (Figure 4D). In this lattice, the disulphide bonds provide diagonal cross-braces, whereas stability in the direction of the unit cell edges is provided by a very extensive interface (∼10 000 Å2), which includes protruding C-terminal hooks, which engage adjacent dodecamers through a series of conserved residues (Figure 1F; Supplementary Figure 1). Figure 5.Inter-subunit disulphide bond. (A) Cartoon diagram of two disulphide-bonded polyhedrin related by a two-fold rotation forming an extended dimer. Structures are coloured by chains and the helices are shown in cylinders. Residues involved in intermolecular salt bridges are drawn as sticks and disulphides are shown as yellow spheres in two orthogonal views. (B) SDS–PAGE of AcMNPV polyhedra under reducing and non-reducing conditions when dissolved in bicarbonate buffer, pH 10.5. Polyhedra were incubated at 80°C for 2 h to inactivate the protease associated with polyhedra before exposure to alkaline buffer. Samples were electrophoresed on 4–8% gradient SDS gels with 1 min (lanes 2–4 and 6) and 5 min (lanes 1 and 5) heating at 100°C. Download figure Download PowerPoint Comparison of the lattice arrangement between baculovirus and CPV polyhedrins Although the protein folds and the interactions, which stabilize the crystal lattice, differ between AcMNPV and CPV (AcMNPV uses disulphide bonds and CPV uses NTPs), there are some broad similarities in the lattice architecture. Not only the size of the unit cell (∼102–103 Å), but also the space groups are identical (I23) and both are assembled from similarly sized trimeric building blocks arrayed on the body diagonals of the cubic lattice. However, the CPV trimers are translated about 6 Å along this diagonal (Figure 2C) and the subunit jelly rolls rotated by ∼137° compared with AcMNPV. Virus occlusion Despite forming an extremely robust lattice, the AcMNPV polyhedra nonetheless harbour pools of flexibility at the crystallographic origin and symmetry-related points (Supplementary Figure 6). These contain the region between residues 32 and 48, which shows the lowest amino-acid sequence conservation across the baculoviruses (Figure 1F; Supplementary Figure 1), suggesting that it may confer specificity for the packaging of a particular virus strain. Baculoviruses, unlike CPVs, do not possess icosahedral symmetry, so there needs to be physical flexibility in the areas, which engage proteins on the surface of the virion envelope, as there is an inevitable symmetry mismatch with the irregular packets of baculoviruses (Figure 4E). This hypothesis is supported by the observation that certain mutations in this region interfere with the packaging