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Journal of Virology, July 2006, p. 6895-6905, Vol. 80, No. 14
0022-538X/06/$08.00+0     doi:10.1128/JVI.00368-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The 2.6-Angstrom Structure of Infectious Bursal Disease Virus-Derived T=1 Particles Reveals New Stabilizing Elements of the Virus Capsid

Damià Garriga,1 Jordi Querol-Audí,1 Fernando Abaitua,4,{dagger} Irene Saugar,3 Joan Pous,2 Núria Verdaguer,1* José R. Castón,3 and José F. Rodriguez4

Institut de Biologia Molecular de Barcelona, CSIC,1 Plataforma Automatizada de Cristalografia PCB-CSIC, Josep Samitier 1-5, 08028 Barcelona,2 Departments of Structure of Macromolecules,3 Molecular and Cell Biology, Centro Nacional de Biotecnología, CSIC, Calle Darwin no. 3, 28049 Madrid, Spain4

Received 22 February 2006/ Accepted 2 May 2006


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ABSTRACT
 
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 Å resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions.


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INTRODUCTION
 
Infectious bursal disease virus (IBDV), the best characterized member of the Birnaviridae family, is an important avian pathogen that inflicts major economic losses to the poultry industry (42). Infection with virulent IBDV strains causes the destruction of pre-B-lymphocyte populations contained within the bursa of Fabricius and, as a consequence, the immunosuppression of affected birds (4).

IBDV possesses a bipartite double-stranded RNA genome encoding five mature polypeptides. The major RNA segment contains two partially overlapping open reading frames (ORFs): the first one codes for the 16-kDa polypeptide VP5 that appears to play an important role in virus dissemination and virulence (28, 44) and the second one codes for the virus polyprotein (110 kDa). The smaller RNA segment contains a single ORF encoding protein VP1, which is the virus RNA-dependent RNA polymerase (11).

The virus polyprotein is cotranslationally processed to give three polypeptides, named pVP2 (54 kDa), VP4 (27 kDa), and VP3 (29 kDa). VP4 is the viral protease responsible for the polyprotein cleavage (23). pVP2 (the VP2 precursor) interacts with VP3, initiating the capsid assembly pathway in which VP3 plays an essential scaffolding role (30). pVP2 is further cleaved at its C-terminal end by an unknown mechanism releasing the mature VP2 (47 kDa). This second processing event requires capsid assembly (6).

The IBDV capsid consists of a single shell formed by 260 trimers of protein VP2 organized in a T=13 icosahedral lattice (2, 5). Previous work aimed at the characterization of the IBDV assembly process revealed that independent expression of the VP2 coding region from chimeric genes leads to the assembly of icosahedral T=1 subviral particles (SVPs) of ~23 nm in diameter, whereas pVP2 expression results in the formation of tubular structures with hexagonal lattices (5). Recently, the crystal structures of the T=13 virion capsids and the T=1 SVP, containing the first 441 VP2 residues from the IBDV vaccine strain CT, have been determined to 7 Å and 3 Å resolution, respectively (7). Both structures confirmed that the building blocks of the IBDV capsids are the VP2 trimers, as was previously anticipated by the cryo-electron microscopy reconstructions (2, 5). The VP2 subunit is folded into three distinct domains named projection (P), shell (S), and base (B). Domains S and P are ß barrels with a jelly roll topology, oriented such that the ß-strands are tangential and radial, respectively, to the spherical particle. The B domain is formed mainly by {alpha}-helices from the N and C termini of the VP2 polypeptide. The first 10 amino acids at the N terminus of VP2 and the last 10 residues at the C terminus were disordered in the T=1 SVPs. However, most of these residues were ordered in some VP2 subunits of the T=13 particles (7).

Here we report the 2.6-Å crystal structure of IBDV, Soroa strain, derived T=1 SVPs. The structure of these particles, containing the first 452 amino acids of pVP2, reveals the presence of two previously unnoticed stabilizing elements of the T=1 particles: (i) a Ca2+ ion located at the threefold icosahedral axis that might act as a sealing element of the VP2 trimers, and (ii) the long C-terminal arm of VP2 that mediates interaction between trimers.


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MATERIALS AND METHODS
 
Generation of transformed Saccharomyces cerevisiae cells expressing the VP2 coding sequence. A DNA fragment corresponding to the region encoding the 452 N-terminal residues of the IBDV polyprotein was generated by PCR synthesis using pVOTE/POLY (15) harboring the complete polyprotein ORF from the IBDV Soroa strain (accession no. AF140705). PCRs were carried out using primers 5'-GCGCAGATCTATGACAAACCTGTCAGATC and 5'GCGCAAGCTTACCTTATGGCCCGGATTATGTCTTTG. The DNA fragment was purified, digested with BglII and HindIII, and cloned into the yeast episomal plasmid vector pESC-URA (Stratagene) previously digested with BamHI and HindIII. The resulting plasmid, pESC-URA/VP2_452, contains the recombinant VP2_452 construct under the control of the inducible Gal1 yeast promoter. pESC-URA/VP2_452 was used to transform the YPH499 (ura3-52 lys2-801amber ade2-101ochre trp1-{Delta}63 his3-{Delta}200 leu2-{Delta}1) S. cerivisiae strain (Stratagene). Transformation, isolation, and maintenance of transformed yeasts were performed according to protocols provided by the manufacturer.

Production and purification of T=1 SVPs. A selected transformed yeast colony was grown in synthetic minimal medium (yeast nitrogen base) (BIO101 Systems) supplemented with complete supplement mixture lacking uracil (BIO101 Systems) and 2% raffinose (Sigma). Cultures were incubated for 24 h at 30°C. Aliquots of these cultures were used to inoculate fresh yeast nitrogen base medium supplemented with complete supplement mixture lacking uracil and 2% galactose (Sigma). Cultures were incubated at 30°C for 16 h. Yeast cells were sedimented by centrifugation (1,000 x g for 5 min at 4°C) and washed twice with distilled H2O. Cell pellets were resuspended in 1 volume of lysis buffer (10 mM Tris [pH 8.0], 150 mM NaCl, and 1 mM EDTA). After adding 1 volume of glass beads (425 to 600 microns) (Sigma), cells were disrupted by vigorous vortexing. The mixture was centrifuged (5,000 x g for 5 min at 4°C). Supernatants were kept at –70°C and eventually used for SVP purification as previously described (40).

Crystallization and data collection. Cubic crystals belonging to space group P213 (a [unit cell parameter] = 326.4 Å) were obtained by the vapor diffusion method in hanging drops at room temperature by mixing equal volumes of VP2 (6 mg/ml) and the reservoir solution containing 12 to 17% PEG 4K. Suitable crystals appeared in these conditions in a pH range between 7.5 and 9.0 in the presence of 3% isopropanol (vol/vol) as an additive.

Crystals were transferred to a cryoprotecting solution containing 20% glycerol in the crystallization buffer and incubated 1 minute before cooling by immersion in liquid nitrogen. A 2.6-Å data set was collected from a single crystal using synchrotron radiation (ESRF, Grenoble, France; beamline ID23-1) with a MarMosaic 225 charge-coupled-device detector. Diffraction images were processed using MOSFLM (26) and internally scaled with SCALA (14) (Table 1).


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  		TABLE 1. Data collection and refinement statistics

Structure determination and refinement. Packing consideration indicated that the crystal asymmetric unit contained one-third of the icosahedral viral particle, or 20 VP2 protomers (Table 1). The three-dimensional structure was determined by molecular replacement with the program AMoRe (36). The atomic coordinates of the closely related SVPs derived from the vaccine strain CT of IBDV, previously determined at 3.0 Å resolution (7) (PDB entry 1WCD), were used as the starting model. An unambiguous single solution for the rotation and the translation functions was obtained, giving an Rfactor of 38.2% and a correlation coefficient of 63%, working with data in the resolution range of 50 to 4.0 Å. At this point, 20-fold noncrystallographic symmetry (ncs) averaging with the DM program (8) was used for phase extension to 2.6 Å. The averaging mask covered the whole asymmetric unit. The procedure resulted in a very clear electron density that allowed the rebuilding of all the differences found between the two structures (Fig. 1). Weak averaged electron density was seen in some of the most prominent surface loops of the IBDV SVPs. In particular, the DE loop of domain S (SDE), around the fivefold axes, and, to a lesser extent, the DE and the HI loops of the protruding domain P (PDE and PHI), appeared partially disordered. Further inspection of the whole asymmetric unit revealed that some of these loops participate in crystal packing interactions. Departures from the strict icosahedral symmetry due to the particle-particle contacts in the crystal resulted in poor averaged maps in those particular regions. To overcome this problem, given the data completeness available (87.8%, in the resolution range 20 to 2.6 Å), the ncs restraints used in refinement were removed in regions involved in packing contacts.


Figure 1
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  		FIG.1. Electron density maps around three different regions of the IBDV-derived T=1 SVP. (A) Stereoscopic view of the C-terminal arm of VP2, residues from 430 to 440, containing the short helix {alpha}4. The 20-fold averaged electron density is shown as a chicken wire diagram in light blue. (B) Stereoscopic view of the acidic cluster and the Ca2+ ion at the threefold axis. Acidic residues Asp31 and Asp174 from the reference molecule are shown in atom-type colors and explicitly labeled; the symmetry-related aspartates are in green and blue, and the Ca2+ ion is shown as a silver ball. The averaged electron density is shown in light blue and the strong peak (4{sigma}) of difference in Fo – Fc electron density is in red. (C) Ribbon diagram showing the different SDE loops in the fivefold axis involved in crystal contacts (left). In the right panel a detail of the sigmaA-weighted 2 Fo – Fc electron density, calculated with REFMAC (35) after removing the ncs restraints in the region, is shown in light blue. The different loops are represented as balls and sticks. The reference loop is in atom-type color and explicitly labeled, and the neighboring loops around the fivefold are in magenta, green, purple, and red.

Refinement was performed by iterative maximum-likelihood positional and translation, libration, and screw-rotation displacement with the REFMAC5 program (35), using tight ncs restraints for most of the VP2 protein. The ncs restraints were removed in loops SDE, PDE, and PHI of VP2 subunits participating in particle-particle interactions. The resulting sigmaA-weighted 2 Fo – Fc and Fo – Fc difference maps calculated with REFMAC (35) allowed the tracing of five different conformations for the SDE loop in the fivefold axes involved in particle-particle contacts (Fig. 1) as well as the rebuilding of the small changes occurring in the main- and side-chain residues of loops PDE and PHI, also involved in crystal packing. Refinement with REFMAC was alternated with manual model rebuilding using the programs TURBO-FRODO (39) and O (22). The final refinement statistics are summarized in Table 1.

Illustrations. Figure 1 was made with the programs Bobscript (13) and Raster 3D (33), and Fig. 2, 4, 5, and 6 were made with PyMol (9).


Figure 2
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  		FIG. 2. The structure of the IBDV-derived T=1 SVPs. (A) Stereoview of a ribbon diagram of VP2. The three different domains P, S, and B are colored orange, blue, and green, respectively. The additional amino acids found at the N and C termini of VP2 are shown in red. The Ca2+ ion is represented as a solid sphere in yellow. All secondary structural elements are explicitly labeled. (B) Stereoscopic view of the C{alpha} trace of the structural superimposition of VP2 determined in this work (red) with the equivalent VP2 structure of the T=1 SVPs (green) and T=13 virion particles (blue) of IBDV (vaccine strain CT) solved by Coulibaly et al. (7). The overall folding of VP2 is closely related in the three structures. The C-terminal extension (amino acids from 431 to 440) found in the T=1 particles in this work, together with the different conformations adopted by the loops SDE and, to a lesser extent, the conformations of the exposed loops PHI (both involved in packing interactions in our structure), constitute the largest differences observed between the VP2 structures. The extra C-terminal arm is explicitly labeled, and the different conformations adopted by the SDE loop are shown in different shades of red. The equivalent SDE loops and the terminal ends in the T=13 particles are shown in various shades of blue. (C) Top view of a VP2 trimer (left). The colors used to show the different domains are the same as in panel A. The Ca2+ ion at the center of the trimer is shown as a yellow sphere. The right panel shows a ribbon diagram of the T=1 particle (with a ~60-Å slab) to illustrate the position of the extra C-terminal arm (red) connecting the different trimers. The positions of one threefold and one fivefold axis of the icosahedral particle are indicated with black symbols.


Figure 4
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  		FIG. 4. The interactions involving the C-terminal arm contribute to the stabilization of the T=1 particles. (A) Ribbon diagram showing the arrangement of three VP2 protomers (shown as green, blue, and red) projecting their C termini towards the threefold axis of a neighboring trimer (gray). The Ca2+ ion, also located on the center of the trimer but in another plane, displaced 20 Å along the threefold axis, has been omitted from the picture for clarity. (B) Main contacts established between the C-terminal helix {alpha}4 (residues from 428 to 440, shown as sticks in green) and the base domain of the adjacent trimer. The interactions take place mainly with helices {alpha}2 and {alpha}3 and the N-terminal T8 of one subunit and with P392 of the other subunit. These regions are shown in a ribbon diagram in gray with residues directly implicated in contacts represented in sticks and explicitly labeled. The C-terminal ends (residues from 437 to 440) of the neighboring {alpha}4-helices are also shown as sticks in red and blue.


Figure 5
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  		FIG. 5. Particle-particle interactions within the crystal. The reference SVPs (in dark blue and gold for the S and P domains, respectively) and the protomers from contacting particles in the crystal are shown in the figure center. The different panels show the details of the interactions established between protomers. The largest network of interactions involves four VP2 protomers contacting the fivefold of the reference particle. Residues directly participating in hydrogen bonds or in hydrophobic contacts are shown as sticks and are explicitly labeled. (A) Residues P222 and G224 of loop PBC, together with residues S315 and Q320 of loop PHI and N279 of loop PFG of the first protomer (green), are hydrogen bonded with main- and side-chain residues of loops SC'C" (residues from G76 to Y80) and S{eta}3{eta}4 (residues T137 and D138) of the reference molecule (dark blue). (B) The second VP2 subunit (yellow) projects the loops PDE (residues R249, V252, and H253), PFG (residue G285), and PHI (residues G318 and D323) to the fivefold axis of the reference SVP, contacting with residue D51 of the loop SBC and with residues from P114 to V117 within the loop SDE from two different VP2 protomers, shown in different shades of blue. (C) The third subunit (blue) contacts the reference SVP through a water-mediated hydrogen bond that bridges D190 (loop PAA') and H253 (loop PDE; gold). This histidine is also hydrogen bonded to Y214 and Q215 within the strand B of the fourth contacting protomer (cyan). In addition, Q215 and S217 are interacting with residues T284 and G285 of loop PFG. (D) The loop PFG of the fourth protomer (cyan) is also interacting with residues Y220 (loop PBC) and S317 and G318 (loop PHI) in the reference particle (gold). (E) The second region of contacts involves interaction between residues S217 of loop PBC and S251 of loop PDE in the reference particle (gold) contacting residues Q320 and Q324 (loop PHI) and residues Y220 and P222 (loop PBC) of a neighboring particle (red), respectively.


Figure 6
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  		FIG. 6. The overall arrangement of the particles in the T=1 and T=13 crystals is related to the spontaneous formation of crystals by the virus in infected cells. (A) A central reference virus (shown as red) is surrounded by six symmetry-related particles in the T=13 (left) and T=1 (right) crystals. The blue box defines the crystallographic unit cell. (B) Ultrathin section of chicken embryo fibroblasts infected with the IBDV Soroa strain. The scale bar represents 500 nm.

Biochemical and electron microscopy analysis of SVP stability. T=1 SVPs assembled in S. cerevisiae cells transformed with plasmid pESC-URA_452 were purified as previously described (5). The sucrose gradient fraction, containing the SVPs, was divided into two aliquots and subjected to dialysis against Tris (10 mM Tris-HCl [pH 8.0], the experiment control) or Tris-EGTA (10 mM Tris-HCl, 5 mM EGTA, pH 8.0) buffer, for 12 h at room temperature. Both samples were then subjected to ultracentrifugation on linear 15 to 40% sucrose gradients prepared either in Tris or Tris-EGTA buffer. The 12 resulting fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and negative-staining electron microscopy as described previously (40).

Ultrathin sectioning of chicken embryo fibroblasts infected with the IBDV Soroa strain, harvested at 48 h postinfection, was performed as described elsewhere (31).

Protein structure accession number. The coordinates and structure factors have been deposited into the Protein Data Bank (accession no. 2GSY).


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RESULTS
 
Quality of the electron density maps. The crystal structure of the IBDV, strain Soroa, derived T=1 SVPs was determined by molecular replacement using 20-fold averaging, starting with the phases of the closely related IBDV (vaccine strain CT) SVPs (7) (Table 1). The resulting map at 2.6 Å resolution allowed recognition of most of the IBDV VP2 side chains without ambiguities. In addition, extra density was found for the positioning of nine new well-ordered residues at the C-terminal end of VP2 and three additional amino acids at the N terminus. The longer VP2 recombinant protein used in this work (452 residues) probably facilitates the folding of the nine additional amino acids, organized in {alpha}-helical conformation, at the C terminus (Fig. 1A). Nevertheless, 7 N-terminal and 11 C-terminal amino acids were disordered. A strong peak of electron density was also seen at the icosahedral threefold axis and was interpreted as a Ca2+ ion tightly coordinating six acidic residues in this region (Fig. 1B). Furthermore, the removal of the ncs restraints in regions that participate in crystal contacts during the final cycles of refinement allowed the tracing of the different conformations for the most flexible loops of VP2, SDE, PDE, and PHI (see Materials and Methods; Fig. 1C).

The final refined asymmetric unit includes 20 copies of VP2 (residues from 8 to 440), with 1 Ca2+ ion per icosahedral threefold axis and 115 well-ordered solvent molecules directly interacting with the VP2 residues. The main-chain conformational angles, calculated with PROCHECK (24), fall into allowed regions of the Ramachandran plot, with 86% of the residues located in the most-favored regions. The root mean square (RMS) deviations of bond lengths and bond angles from ideal geometry are shown in Table 1.

New features stabilizing the IBDV-derived SVPs. The structure of the T=1 particles determined in this report is, as expected, very similar to that described for the SVPs derived from the closely related vaccine strain CT, as determined recently (7). The conformation and spatial disposition of the three VP2 domains, B, S, and P, are well preserved in both structures (Fig. 2A). The RMS deviation for the superimposition of C{alpha} atoms from 420 structurally equivalent residues in the two viruses is only 0.41 Å. The main differences between the two structures are found at the innermost region of the B domain and at the threefold and fivefold icosahedral axes (Fig. 2B and C).

As described previously (7), the building block for virus assembly is the VP2 trimer, and 20 trimers interacting via icosahedral twofold and fivefold contacts form the T=1 SVP. All three VP2 domains participate extensively in inter- and intratrimer contacts. In addition to the previously described interactions, the 2.6-Å structure determined in this work reveals the presence of two new elements that contribute to the stabilization of the T=1 particles: (i) a metal ion at the threefold axis and (ii) the C-terminal helix {alpha}4 (Fig. 1A, 1B, 2A, and 2C).

(i) Stability mediated by Ca2+ ions. The Ca2+ ion located on the threefold axis of each VP2 trimer strongly coordinates a cluster of six acidic residues in a perfect octahedral geometry (Fig. 1B). The amino acids involved in interactions with the metal ion are the residues Asp31 and Asp174, both in domain S (Fig. 1A), and counterparts from adjacent threefold related VP2 copies.

In order to analyze the possible contribution of Ca2+ ions to SVP stability, a suspension of purified SVPs was dialyzed against EGTA (see Materials and Methods) and then analyzed by sucrose gradient sedimentation and negative-staining electron microscopy. In fact, EGTA-treated SVPs exhibited an abnormal migration on the sucrose density gradients compared to control samples (Fig. 3A). The comparative electron microscopy analysis of the gradient fractions containing VP2 revealed that while control samples contained abundant SVPs with a typical T=1 morphology, most SVPs were dissociated after EGTA treatment (Fig. 3B).


Figure 3
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  		FIG. 3. T=1 capsids are stabilized by Ca2+ ions. (A) T=1 SVPs in Tris buffer (top) or dialyzed against Tris-EGTA buffer (bottom) were subjected to ultracentrifugation on 15 to 40% sucrose density gradients prepared either in Tris or Tris-EGTA; 12 fractions (lanes F1 through F12) were collected and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blotting with anti-VP2 antibodies. (B) Electron microscopy analysis of fraction 8 of T=1 capsids maintained in either Tris (left) or Tris-EGTA buffer (right). The scale bar represents 100 nm.

(ii) Stability mediated by the helix {alpha}4. The C terminus of VP2 is projected away from the protein core, oriented towards the threefold axis of the neighboring VP2 trimer (Fig. 4A). The short helix {alpha}4 at the end of this region is located below the base domains of the neighboring trimer, interacting mainly with residues in helices {alpha}2 and {alpha}3 and with the N-terminal end of one subunit, and, to a lesser extent, with the loop connecting the 310 helix {eta}7 of the adjacent subunit in the trimer (Fig. 4B). In addition, each {alpha}4-helix contacts the other homologous helices at the threefold axes (Fig. 4A and B).

Particle-particle interactions in crystal packing. Evaluation of the IBDV SVP-SVP contacts in our P213 crystals (Fig. 5) revealed two different interacting regions leading the crystal packing:

(i) The first region includes the exposed loops (PBC, PDE, PFG, PHI, and PAA'; Fig. 2) of four VP2 subunits from two different trimers in one SVP that contact residues around the fivefold axis of the neighboring particle (loops SBC, SDE, SC'C", and S{eta}3{eta}4; Fig. 2). The contact interface is large, with at least 37 amino acids involved in interactions (Fig. 5, boxes A to D). These interactions are mainly responsible for the departure of the icosahedral symmetry observed (see Materials and Methods) as a consequence of the important structural changes occurring in the conformations of loop SDE (Fig. 1C and 2B) in 3 of the 12 icosahedral fivefold axes of the particle and, to a lesser extent, to the changes occurring in the main chain of loops PHI and PFG and in the conformation of the side chains of residue His253 of loop PDE in the VP2 P domains participating in particle-particle interactions.

(ii) The second region of contacts is small and involves only residues Ser117 of strand PB and Ser251 of loop PDE interacting with residues of loops PHI and PBC of a neighboring particle, respectively (Fig. 5, box E).

It is interesting to note that amino acid residues at position 253 (PDE loop) and 284 (PFG loop), determinants of virulence in IBDV (3, 43), are all participating in particle-particle contacts (Fig. 5).


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DISCUSSION
 
The role of the Ca2+ ions and the VP2 C terminus in capsid assembly. As described previously by Coulibaly et al. (7), all three domains of VP2 participate in VP2-VP2 contacts to stabilize the VP2 trimers as the building blocks of the IBDV capsid assemblies. Besides the interactions described by these authors, the Ca2+ ion occupying the threefold axis of the T=1 particle determined in this work (Fig. 1B) plays an additional role in the maintenance of this trimeric structure, acting as glue that sticks together the three subunits through the tight contacts with the side chains of six aspartic acid residues. The presence of cations on the threefold and fivefold icosahedral axes has been described elsewhere in different viruses (1, 18, 38, 41). In all these structures, the divalent cations seem to be involved in the regulation of capsid stability. The critical role of Ca2+ ions in the stability of the IBDV SVPs has been clearly demonstrated by biochemical and electron microscopy analysis (Fig. 3).

The newly described ordered region of the VP2 C-terminal end, determined in this work, and in particular, the short helix {alpha}4 appear to function as an additional element further stabilizing the T=1 SVP structure by producing a long range of interactions throughout the icosahedral threefold axes. In the structure determined, each trimer binds three {alpha}4-helices from a set of three neighboring trimers, and its own {alpha}4-helices are bound by a different set of three neighboring trimers (Fig. 4).

Interactions between {alpha}4-helices around the three folds define a fixed angle between S domains of neighboring trimers that leads to a pentameric grouping of trimers and, finally, to a T=1 icosahedral capsid. In the T=13 capsids of IBDV virions, the VP2 trimers are grouped not only in pentamers but also in hexamers. The existence of different trimer-trimer interfaces in the T=13 capsid (7) implies differences in the angles between neighbor trimers. These subtle differences, required to build a capsid T > 1, may be controlled by flexible regions in the protein (loops, N- or C-terminal ends), duplex or single-stranded RNA, metal ions, or different combinations of these elements (21). In the process of IBDV capsid assembly, a molecular switch has been identified at the region 443 to 452 of the precursor pVP2, which is located just C-terminal to the {alpha}4-helix. This region that is disordered in the present structure appears to be organized in an amphiphilic {alpha}-helix in the virion and would interact with the C-terminal end of VP3 that acts as a scaffold leading the T=13 organization (40).

In light of the observed interactions, mediated by the preceding {alpha}4-helix, it is tempting to suggest a temporal role for the {alpha}4-helix maintaining the curvature of the fivefolds during the T=13 capsid assembly.

Crystal packing contacts involve regions containing residues related to virulence. A characteristic feature of IBDV-infected cells is the presence of viroplasms or inclusion bodies (IB) with a distinctive honeycomb appearance within the cell cytoplasm. These aggregates, formed by large numbers (from hundreds to thousands) of closely packaged virus particles, are usually referred to as paracrystalline IBDV arrays (29). During the final step of the infectious cycle, IBDV-infected cells are lysed, thus releasing IB to the extracellular medium (Fig. 6). One of the major problems in controlling IBDV dissemination is the extreme resistance of the virus to environmental conditions and virucidal agents (12). Despite their possible contribution to virus dissemination, IBDV IB have received very little attention. Viruses of different origins use inclusion bodies as part of their overall transmission strategies. A good example of this is the cytoplasmic polyhedrosis virus (genus Cypovirus). This virus has a single T=2 capsid layer occluded within large IB proteinaceous polyhedra (19). These IB are dissolved in the insect alkaline midgut, releasing infectious virions (20). IBDV, with a single T=13 capsid layer, might assemble into rigid paracrystalline arrays to favor the integrity of the innermost particles against adverse conditions and/or virus neutralizing antibodies. Free virions are also present in the cytoplasm and would be important for cell-cell dissemination. The three-dimensional organization of IBDV IB, as observed in thin sections by electron microscopy, seems to be related with the three-dimensional arrangement observed not only in our crystals but also in crystals of intact virus (Fig. 6B). As mentioned in the Results section, the VP2 amino acids at positions 253 (loop PDE) and 284 (loop PFG) involved in IBDV adaptation to tissue culture growth (27, 34) and virulence (3, 43), located at the most exposed loops of domain P, directly participate in particle-particle contacts (Fig. 5) in crystal packing. The inspection of the crystal packing in the previously published structure of the T=13 virions (1WCE) also reveals the key participation of the exposed loop PDE in virus-virus contacts. Interestingly, these residues participate neither in the VP2 fold nor in interactions required to stabilize the virion. These observations suggest that the ability of the virus to assemble into paracrystalline IB might be related to virulence. The characterization of IBDV isolates harboring mutations of amino acid residues specifically involved in particle-particle contacts might help in assessing this hypothesis.

The fivefold axis; implications in mRNA translocation. At the interior of the particles, the {alpha}3-helices of VP2 domain B are organized around fivefold axes forming a pentameric channel wide enough (diameter, ~20 Å) for the extrusion of single-stranded RNA molecules. The flexible loop SDE at the external surface reaches the fivefold axes sealing this channel (Fig. 7). The size and shape of the pentameric channel in the T=1 particles is similar to that of the native T=13 virions. An inspection of the electrostatic potential at the interior surface of the fivefold axis (Fig. 7) showed that the channel is mostly electronegative but contains small electropositive regions and a ring of hydrophilic amino acids at the outer region of the channel. The electronegative nature of the channel would facilitate the extrusion of the newly transcribed mRNA molecules, traversing by floating away from the repulsive walls of the channel. Similar situations were found in the fivefold axes of the rotavirus middle shell (32) and in other proteins involved in translocation of nucleic acids as bacteriophage connectors or the bacterial conjugation protein TrwB (16, 17). The extrusion of mRNAs through pores at the fivefold axes has been previously observed for members of the Reoviridae family (10, 25). Finally, the flexible SDE loop that seals the fivefold channels at the vertices of the particle could possibly display a concerted switch mechanism between different conformations, serving as a gate for mRNA translocation.


Figure 7
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  		FIG. 7. The interior of the fivefold channel. The molecular surface of a pentamer subunit is shown as inside (A) and side (B) views. Surfaces are represented with the electrostatic potential calculated with the program GRASP (37) in blue and red for positive and negative charges, respectively. A single-stranded RNA (green) traversing the channel has been modeled in panel B.


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ACKNOWLEDGMENTS
 
Work in Barcelona was supported by grants BIO2002-00517 and BFU2005-02376/BMC. Work in Madrid was supported by grant AGL2003-07189 from Dirección General de Investigación del Ministerio de Educación. D.G. acknowledges the I3P fellowship from CSIC.

X-ray data were collected at the EMBL protein crystallography beam line ID23.1 at ESRF (Grenoble) within a Block Allocation Group (BAG Barcelona). Financial support was provided by the ESRF.

We are indebted to I. Fita and D. Blaas for critically reading the manuscript and to E. Campanario for her technical advice on S. cerevisiae fermentation.


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FOOTNOTES
 
* Corresponding author. Mailing address: IBMB-CSIC Parc Cientific de Barcelona, Josep Samitier 1-5, 08028 Barcelona, Spain. Phone: 34 93 403 49 52. Fax: 34 93 403 49 79. E-mail: nvmcri{at}ibmb.csic.es. Back

{dagger} Present address: Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom. Back


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Journal of Virology, July 2006, p. 6895-6905, Vol. 80, No. 14
0022-538X/06/$08.00+0     doi:10.1128/JVI.00368-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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