Crystal Structure of the Avian Reovirus Inner Capsid Protein {sigma}A

Avian reovirus, an important avian pathogen, expresses eightstructural and four nonstructural proteins. The structural ${sigma}$ Aprotein is a major component of the inner capsid, clamping together ${lambda}$ A building blocks. ${sigma}$ A has also been implicated in the resistanceof avian reovirus to the antiviral action of interferon by stronglybinding double-stranded RNA in the host cell cytoplasm and thusinhibiting activation of the double-stranded RNA-dependent proteinkinase. We have solved the structure of bacterially expressed ${sigma}$ A by molecular replacement and refined it using data to 2.3-Åresolution. Twelve ${sigma}$ A molecules are present in the P1 unit cell,arranged as two short double helical hexamers. A positivelycharged patch is apparent on the surface of ${sigma}$ A on the insideof this helix and mutation of either of two key arginine residues(Arg155 and Arg273) within this patch abolishes double-strandedRNA binding. The structural data, together with gel shift assay,electron microscopy, and sedimentation velocity centrifugationresults, provide evidence for cooperative binding of ${sigma}$ A to double-strandedRNA. The minimal length of double-stranded RNA required for ${sigma}$ A binding was observed to be 14 to 18 bp.

INTRODUCTION

Top

INTRODUCTION

Avian reovirus belongs to the Orthoreovirus genus of the Reoviridaefamily. It has been linked to various diseases in birds, whichgenerate considerable economic losses in the poultry industry(17). The virus has a double concentric icosahedral capsid (2).The inner capsid is made up of the ${lambda}$ A, ${lambda}$ C, and ${sigma}$ A proteins, whichencapsulate a double-stranded RNA (dsRNA) genome consistingof 10 segments and up to 12 copies of the RNA-dependent RNApolymerase. The RNA polymerase consists of proteins ${lambda}$ B and µA.Proteins µB, ${sigma}$ B, and ${sigma}$ C are components of the outer shell.The virus also encodes four nonstructural proteins (2), whichare involved in cell fusion (p10), viral assembly (µNSand ${sigma}$ NS), or as-yet-undetermined functions (p17). Up to now,only the crystal structure of the primary cell attachment protein ${sigma}$ C has been reported (14).

The object of our present study, the protein ${sigma}$ A, has two functionsin avian reovirus biology. On one hand, it plays a structuralrole, forming part of the inner capsid. In this respect it isthe structural equivalent of mammalian reovirus ${sigma}$ 2. The structureof the mammalian reovirus inner capsid has been solved by X-raycrystallography (33). ${sigma}$ 2 is present in 150 copies per core, inthree different locations ( ${sigma}$ 2-i, ${sigma}$ 2-ii, and ${sigma}$ 2-iii), acting asa stabilizing clamp on the outside of the inner capsid. Theprotein interacts extensively with ${lambda}$ 1 (the mammalian reovirusequivalent of ${lambda}$ A) at two different binding sites (the bindingsites on ${lambda}$ 1 for ${sigma}$ 2-ii and ${sigma}$ 2-iii are similar). There are only sparsecontacts between ${sigma}$ 2 monomers and between ${sigma}$ 2 and the other coreprotein ${lambda}$ 2 (the equivalent of avian reovirus ${lambda}$ C). Cryo-electronmicroscopy of avian reovirions confirmed that avian reoviruscontains 150 ${sigma}$ A "nodules" contacting mainly ${lambda}$ A, with minor contactsformed with the ${lambda}$ C turrets and other ${sigma}$ A molecules (40). ${sigma}$ A alsocontacts the µB proteins of the outer capsid (equivalentto µ1 in mammalian reovirus).

Apart from its structural role in the virus, ${sigma}$ A has been shownto strongly bind dsRNA, and in this way it has been implicatedin the virus's resistance to interferon by preventing the activationof the dsRNA-dependent protein kinase (PKR) (13, 25). This interactionwould presumably take place in the host cell cytoplasm (outsidethe virus). Here, ${sigma}$ A appears to play a role analogous to thatof the mammalian reovirus outer capsid protein ${sigma}$ 3, as this protein(the structural equivalent of avian reovirus ${sigma}$ B) also exhibitsdsRNA binding activity (39), albeit not as strongly as avianreovirus ${sigma}$ A (25). Here we report the crystal structure of recombinant ${sigma}$ A and show that it self-assembles in a helical fashion. Mutationalstudies support the hypothesis that dsRNA may bind to the insideof this helix.

MATERIALS AND METHODS

Top

MATERIALS AND METHODS

For structural studies, the avian reovirus S1133 protein ${sigma}$ A wasexpressed as a maltose binding protein fusion, cleaved usingthe protease factor Xa, purified, and crystallized as describedpreviously (15). Data collection, processing, and scaling wereperformed as described in the same paper. Molecular replacementwas performed using a model based on the structure of mammalianreovirus ${sigma}$ 2 (33). To construct the model used in molecular replacement,alignment of the ${sigma}$ A and ${sigma}$ 2 sequences was performed with CLUSTALW(4); this alignment and the ${sigma}$ 2 structure were input into theprogram CHAINSAW (6), which retains conserved amino acids butprunes nonconserved residues to the C-gamma atom. Molecularreplacement itself was performed with MOLREP (35), using theoption that adapts the search model by recalculating the temperaturefactor of each atom as a function of its surface area. Modeladjustment was done using COOT (11) and O (18), refinement wasperformed with REFMAC (27), and water molecules were built usingARP (23). Model validation was performed with PROCHECK (24),WHATCHECK (36), MOLPROBITY (7), and MOLEMAN (19).

Site-directed mutagenesis was performed using the QuikChangekit (Stratagene, Cultek, Madrid, Spain), following the manufacturer'sinstructions. ${sigma}$ A and mutated ${sigma}$ A were purified as described previously(15). Gel shift assays were performed using ${sigma}$ A at 0.6 mg/ml inTE buffer (10 mM Tris-HCl, pH 8.5, 1 mM EDTA [disodium EDTA])and a dsRNA ladder which contains seven dsRNAs of differentlengths (500, 300, 150, 80, 50, 30, and 21 bp) at 0.5 mg/ml(New England Biolabs, Izasa S.A., Barcelona, Spain). Increasingamounts of ${sigma}$ A (0, 0.6, 1.2, 3.0, and 6.0 µg) were incubatedwith 0.5 µg of dsRNA ladder in TE buffer-150 mM sodiumchloride for 10 min on ice, and then samples were analyzed bynondenaturing polyacrylamide gel electrophoresis (see the legendto Fig. 3B for details). The dsRNA was stained with ethidiumbromide and visualized under UV light. To determine the minimumdsRNA length that ${sigma}$ A can bind, gel shift assays were performedusing ${sigma}$ A in phosphate-buffered saline (PBS) buffer (137 mM sodiumchloride, 2.7 mM potassium chloride, 8 mM disodium hydrogenphosphate, 15 mM potassium dihydrogen phosphate, pH 7.4), supplementedwith 2 mM magnesium chloride and three dsRNAs of different lengths(18, 14, and 10 bp). Experiments were performed in which increasingconcentrations of ${sigma}$ A (0, 16.0, and 38 µM) were added tofixed concentrations of dsRNA (1.6 µM of 18-bp dsRNA,3.3 µM of 14-bp dsRNA, and 3.6 µM of 10-bp dsRNA)in a final volume of 12.2 µl leading to ${sigma}$ A-to-dsRNA ratiosindicated in the figures. Mixtures were incubated for 10 minat room temperature and analyzed on a 15% polyacrylamide gelin TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA,pH 8.0). The dsRNA was visualized as described above.

View larger version (50K):
[in this window]
[in a new window]

FIG. 3. dsRNA binding of the ${sigma}$ A protein. (A) Electrostatic surface of ${sigma}$ A. On the left the orientation is as in Fig. 1A; on the right the view has been rotated 180° around a vertical axis centered on the apex. Positively charged residues on the molecular surface are labeled. The location of Gln305 is also shown, whereas Arg38 is shown in parentheses, as it is barely surface exposed. (B) dsRNA gel shift assays. The assays were performed using ${sigma}$ A at 0.6 mg/ml in TE buffer (10 mM Tris-HCl, pH 8.5, 1 mM EDTA) and a dsRNA ladder which contains seven types of dsRNA of different lengths (500, 300, 150, 80, 50, 30, and 21 bp) at 0.5 mg/ml. Increasing amounts of ${sigma}$ A (0, 0.6, 1.2, 3.0, and 6.0 µg) were incubated with 0.5 µg of dsRNA ladder in TE buffer-150 mM NaCl for 10 min on ice. Then, samples were analyzed by nondenaturing gel electrophoresis. (Left) A 10% polyacrylamide gel in TBE buffer was used. (Middle and right) Discontinuous gels as described by Laemmli (22) were used, with a 10% polyacrylamide separating gel and omission of sodium dodecyl sulfate from the sample, the gel, and the running buffer (13). Nonmutated ${sigma}$ A protein was used in the left and middle panels. The right panel shows a gel shift assay performed with the Arg155-to-Ala mutant. Identical results were obtained with the Arg273-to-Ala mutant (data not shown). An arrow in the middle panel indicates the high-molecular-weight complexes observed. (C) Binding of ${sigma}$ A to small dsRNA fragments as observed by gel shift assay. In the first three lanes an 18-bp dsRNA was used, in the next three lanes a 14-bp dsRNA was used, and in lanes 7 to 9 a 10-bp dsRNA was used. In lane 10 ${sigma}$ A alone was loaded. ${sigma}$ A/dsRNA ratios are indicated above the lanes.

Sedimentation velocity analysis was carried out with a BeckmanOptima XL-A analytical ultracentrifuge equipped with absorbanceoptics, using an An-50Ti rotor and standard 12-mm optical pathdouble-sector centerpieces of Epon-charcoal. ${sigma}$ A protein was preparedas described before; an RNA oligonucleotide with the sequence5'-ACA CUG UGA UCA GCA GGC GCC AGC UGA UCA CAG UGU-3' was obtainedfrom Nedken (Barcelona, Spain). dsRNA was prepared from thisself-complementary oligonucleotide by heating and slow cooling.We used PBS buffer for all experiments, supplemented with 2mM magnesium chloride. Samples of 0.4 ml with ${sigma}$ A and dsRNA atdifferent ratios were centrifuged at 40,000 rpm and 20°C.The concentration of ${sigma}$ A in samples with excess protein was 4.9µM, while dsRNA was added at 0.0, 1.0, 2.0, and 3.0 µM(duplex concentration), leading to ${sigma}$ A-to-dsRNA ratios of 1:0.0,1:0.21, 1:0.41, and 1:0.62, respectively. The concentrationof dsRNA in samples with excess dsRNA was 1.77 µM (duplexconcentration), while ${sigma}$ A was added at 0.0, 1.71, 0.58, 0.18,and 0.058 µM, leading to ${sigma}$ A-to-dsRNA ratios of 0:1.0, 1:1.0,1:3.1, 1:9.3, and 1:31, respectively. For calculation of theconcentrations, the calculated molar extinction coefficientsof ${sigma}$ A and the dsRNA based on their sequence were used (95,590M^–1 cm^–1 at 280 nm for ${sigma}$ A, 347,100 M^–1 cm^–1at 260 nm for the dsRNA). Radial absorbance scans were takenat 280 nm (experiment 1) or 243 nm (experiment 2), and datawere analyzed using the c(s) continuous distribution of Lammequation solutions with the software SEDFIT (34).

Theoretical values of the sedimentation coefficient s_20,w wereestimated with the HYDROPRO program (12), using models composedof one, two, or three copies of chain G of our ${sigma}$ A structure anda model of canonical A-form dsRNA of the sequence used. ${sigma}$ A moleculeswere placed in the center of the RNA. The partial specific volumefor ${sigma}$ A (0.728 ml/g) was calculated from its amino acid compositionautomatically by HYDROPRO. For dsRNA a value of 0.55 ml/g wasused. For the different complexes, weight-averaged values wereemployed, giving 0.669, 0.692, and 0.792 for the putative 1:1,2:1, and 3:1 ${sigma}$ A-dsRNA complexes, respectively. As recommendedin the HYDROPRO manual, values of 3.2, 2.8, and 3.0 were usedfor the effective atomic radii of protein, dsRNA, and protein/dsRNAsamples, respectively.

For electron microscopy, ${sigma}$ A alone at 0.05 mg/ml and mixturesof ${sigma}$ A with dsRNA (0.05 mg/ml ${sigma}$ A; 1.8 µM 36-bp dsRNA; molarratio, 15:1) in PBS buffer plus 2 mM magnesium chloride wereapplied to the clean side of carbon on mica (carbon/mica interface)and negatively stained with 1% (wt/vol) sodium silicotungstate(pH 7.0). Micrographs were taken under low-dose conditions witha JEOL 120 EX II microscope at 100 kV and a nominal magnificationof x40,000.

Figures 1A and B, 2A, and 4 were prepared using Pymol (PyMOLMolecular Graphics System, DeLano Scientific, San Carlos, CA).Figure 3A was prepared using the program CCP4 mg (31).

View larger version (38K):
[in this window]
[in a new window]

FIG. 1. Structure of the avian reovirus ${sigma}$ A protein. (A) Stereo view from the top of the ${sigma}$ A pyramid (ribbon representation). The monomer with the lowest average temperature factor, G, was used to make this figure. The amino and carboxy termini of the protein are labeled. (B) Stereo view of main chain superimposition of all 12 ${sigma}$ A copies in the asymmetric unit of the crystal. The amino-terminal Met, carboxy-terminal Ala, and every 50th residue are labeled, as are the amino acids flanking the disordered loop (Gln37 and Ile61). (C) Topology of the ${sigma}$ A protein chain. Alpha-helices are shown as rectangles labeled with numbers, and beta-strands are shown as arrows labeled with letters. The two structurally homologous domains 1 and 2 are shown in orange and blue, and the carboxy-terminal domain 3 is shown in green. Helix 2 (in white) is not observed in our structure but is inferred from the structure of mammalian reovirus ${sigma}$ 2 (see text). Starting and ending residues of the secondary structure elements are labeled.

View larger version (45K):
[in this window]
[in a new window]

FIG. 2. Comparison of avian reovirus ${sigma}$ A structure with mammalian reovirus ${sigma}$ 2. (A) Superposition of ${sigma}$ A onto ${sigma}$ 2. ${sigma}$ A chain G was superposed on ${sigma}$ 2 chains D and E (PDB entry 1EJ6 [33]), leading to an overall RMSD of 1.6 Å for 390 or 392 superimposed C-alpha atoms, respectively (20). ${sigma}$ A is shown in black, ${sigma}$ 2 chain D is shown in dark gray, and ${sigma}$ 2 chain E is shown in light gray. (B) Structure-based alignment of ${sigma}$ A and ${sigma}$ 2. Secondary structure elements in ${sigma}$ A are indicated (S for strand, H for helix) and numbered.

View larger version (79K):
[in this window]
[in a new window]

FIG. 4. Assembly of ${sigma}$ A molecules in the crystal and model of dsRNA binding. (A) Stereogram of the dodecameric assembly of ${sigma}$ A in the crystallographic asymmetric unit. Monomers are shown in cartoon format and colored differently (two yellow, two orange, two red, two green, two blue, one magenta, and one cyan). (B) Model of a ${sigma}$ A trimer interacting with a 36-bp canonical A-form dsRNA (yellow). Residues implicated in RNA binding, Arg155 and Arg273, are shown in red; Gln305 is shown in magenta.

Protein structure accession number. The coordinates have been deposited in the protein structuredatabase (http://www.rcsb.org) under accession code 2VAK; thestructure factors are also available.

RESULTS

Top

RESULTS

Bacterial expression, purification, and crystallization of the ${sigma}$ A protein have been described previously (15). Molecular replacementyielded a solution with 12 copies of a model based on the mammalianreovirus core protein ${sigma}$ 2. The ${sigma}$ A molecules occupy 38% of the unitcell (Matthews coefficient 3.2 [26]). The final refinement wasperformed without using noncrystallographic symmetry restraints;statistics are shown in Table 1. The final model contains aminoacids Met1-Gln37 and Ile51-Ala416 of ${sigma}$ A for each of the 12 chains,12 sulfate ions, and 3,003 water molecules. Some monomers alsocontain a vestige of the expression linker segment remainingafter factor Xa protease cleavage (Ile-Ser-Glu-Phe-Gly-Ser-Thr),whereas in others this segment is partially or completely disordered.The conformation of the expression tail will not be furtherdiscussed here and is not included in the analyses describedbelow.

View this table:
[in this window]
[in a new window]

TABLE 1. Refinement statistics

Structure and topology of ${sigma}$ A. The overall topology of the ${sigma}$ A monomer is roughly tetrahedralin form with an approximately rectangular base (Fig. 1). Thebase is relatively planar, 65 to 70 Å long and 45 to 50Å wide, and encompasses the proposed site of interactionwith the main core protein ${lambda}$ A (see below). The distance fromthe base to the apex, which is expected to form the interactionsite with µB in the mature virion, is around 45 Å.The 12 crystallographically independent copies in the unit cellare virtually identical (root mean square deviation [RMSD] ofC-alpha atoms after superimposition of 0.2 to 0.7 Å; Fig.1B). Each ${sigma}$ A monomer contains 12 alpha-helices, one mixed three-strandedbeta-sheet, and three antiparallel two-stranded beta-sheets(Fig. 1C).

There is a gap where the protein chain is disordered in all12 copies present in the crystal between helices 1 and 3. Nointerpretable electron density was observed for ${sigma}$ A residues 38to 50 in this region. In ${sigma}$ 2, the structurally equivalent aminoacids are 39 to 52 (Fig. 2). In the previously solved mammalianreovirus core structure, this region contains an alpha-helixin one of the ${sigma}$ 2 copies (residues 39 to 46) which is melted inthe other two (33). This region contacts ${lambda}$ 1 in the mammalianreovirus core (33), so it is conceivable that the ${sigma}$ A alpha-helixbecomes ordered only upon binding to ${lambda}$ A. We have designated theputative equivalent helix in ${sigma}$ A as helix 2. The fact that thisloop is flexible and can adopt various conformations suggeststhat it may be used by the protein to adapt to the differentbinding environments that the ${lambda}$ A shell presents.

The N and C termini are both oriented toward the apex of themolecule and are solvent accessible in this context (althoughin the virion they are likely to be buried in the interfacewith µB). The first secondary structure element in theN-terminal region, beta-strand A, however, is buried in thecore of the molecule. Phe-8 and Phe-9 in this strand both packagainst surrounding hydrophobic and aromatic residues. Helices1, 3, and 4 (and the putative helix 2 [see below]), togetherwith beta-sheets B-C and A-D, form a discrete subdomain, whichis packed against another subdomain with the same topology (helices5, 6, 7, and 8 and sheets G-F and H-E). These two domains arepacked against a third domain formed by helices 9, 10, and 11and beta-strand I (which lies alongside strand E, forming theH-E-I beta-sheet motif). The apex of the pyramid is largelyformed by the loop between helix 8 and strand I.

The regions of ${sigma}$ A contacting ${lambda}$ A and µB can be predictedfrom the previously published crystal structure of the mammalianreovirus core (PDB code 1EJ6 [33]), the published pseudoatomicmodel of the entire mammalian reovirus obtained by fitting knowncrystal structures into cryo-electron microscopic density (PDBcode 2CSE [41]), and cryo-electron microscopic studies of avianreovirus (40). The interaction site with µB mainly comprisesresidues contained within this third domain, while the proposed ${lambda}$ A binding site is almost exclusively composed of residues fromdomains 1 and 2. Domains 1 and 2 present the same secondarystructural elements on the surface at the ${lambda}$ A binding site, potentiallyindicating domain duplication, where the two domains have evolvedto interact with different facets of the ${lambda}$ A component in theinner shell.

Structural and functional comparison with other proteins. Analysis of the whole structure, or domains 1, 2, or 3 independently,by three-dimensional secondary structure matching (20) did notreveal any close structural homologues for ${sigma}$ A, other than mammalianreovirus ${sigma}$ 2, with which it shares 29% sequence identity. Theoverall topologies of these proteins are very similar; however,there are small differences in some surface residues, whichmay be related to inherent flexibility in these regions (Fig.2A). Most of the surface residues that differ between ${sigma}$ A and ${sigma}$ 2 vary in position between the 12 independent ${sigma}$ A monomers inthe unit cell (Fig. 1B and 2A). One notable difference between ${sigma}$ A and ${sigma}$ 2 occurs at the carboxy terminus of helix 10 (residues374 to 388 in the ${sigma}$ A structure), which is markedly distortedin ${sigma}$ A.

dsRNA binding of ${sigma}$ A. With regard to dsRNA binding, ${sigma}$ A is functionally, though notstructurally, analogous to mammalian reovirus ${sigma}$ 3. ${sigma}$ A and ${sigma}$ 3 bothbind dsRNA and have been implicated in PKR inhibition (13, 16).As with ${sigma}$ A, the ability of ${sigma}$ 3 to bind dsRNA has been linked toits capacity to inhibit dsRNA-dependent activation of PKR throughcompetitive binding. It has been proposed that ${sigma}$ 3 binds dsRNAas a dimer and that multiple dimers may line up along the RNAmolecule with their twofold axes aligned along the RNA localdyads (30).

When the electrostatic potential is superimposed onto the surfaceof the ${sigma}$ A monomer, a positively charged region, containing amongother residues Arg155 and Arg273, is notable (Fig. 3A). We mutatedthese residues independently to alanine and examined the effectson dsRNA binding by gel shift assays (Fig. 3B). Both mutationsabolished the dsRNA binding properties of ${sigma}$ A (shown for the Arg155-to-Alamutation), whereas mutation of Arg134, Arg135, Arg389, or Arg390had no significant effect on dsRNA binding. Both mutant proteinswere soluble, and there were no indications that either of themutations affected the protein stability or had any structuralimpact. A further indication that this region may play a rolein RNA binding comes from the binding of a sulfate ion at ornear Gln305, which is close to Arg155 and Arg273. This sulfateis present in each of the 12 monomers in our structure and maymimic the binding of phosphate in the RNA backbone at this site.

In the gel shift assays using native ${sigma}$ A, high-molecular-weightcomplexes were observed that barely enter the stacking gel (Fig.3B, middle panel). This suggests either that ${sigma}$ A and dsRNA mayform large complexes or that the resulting complex may be relativelyuncharged at the pH employed here (8.0). The latter is in keepingwith a model where ${sigma}$ A molecules (theoretical pI 8.6) cover thebulk of the surface of the dsRNA, thus masking negative chargesarising from the phosphate backbone. The dsRNA-binding experimentsalso indicate that ${sigma}$ A is able to bind RNA down to a minimum lengthof at least 21 bp (Fig. 3B, left panel). However, as dsRNA sizeincreases, complexes form at lower ${sigma}$ A concentrations, which suggeststhat the binding is cooperative.

To identify the minimal length of dsRNA bound by ${sigma}$ A, gel shiftassays were performed with 10-bp, 14-bp, and 18-bp dsRNAs (Fig.3C). This showed that ${sigma}$ A can bind 18-bp dsRNA and also 14-bpdsRNA, albeit less efficiently, while 10-bp dsRNA was not boundby ${sigma}$ A. In contrast, ${sigma}$ 3 has been reported to require a minimumlength of 32 to 45 bp for efficient RNA binding (39). We investigatedwhether the putative dsRNA binding site is structurally relatedto the dsRNA binding motif (dsRBM) identified in several otherproteins (3), including PKR (28, 29). The archetypal dsRBM consistsof a contiguous alpha-beta-beta-beta-alpha fold, in which thetwo helices are arranged on one side, packed against a three-strandedantiparallel beta-sheet. The residues implicated in dsRNA interactionare located in loop 2 (between beta-strands 1 and 2) and loop4 (between beta-strand 3 and alpha-helix 2). The dsRNA bindingsite of ${sigma}$ A is somewhat similar and is made up from a discontinuousalpha-beta-beta-beta-alpha subdomain, in which helix 11 andhelix 4 and/or helix 8 lie on opposite sides of a three-strandedmixed beta-sheet. The arginines implicated in dsRNA interactionare located in the loop between alpha-helix 4 and beta-strandE and in the loop between alpha-helix 8 and beta-strand H. However,these loops are not conserved and have a different connectivityin the dsRBM, suggesting that the structural similarity is superficial,and hence that there is no direct evolutionary relationshipbetween the ${sigma}$ A dsRNA-binding region and the dsRBM.

Interactions between ${sigma}$ A molecules in the crystal. ${sigma}$ A is monomeric in salt solutions as judged by size-exclusionchromatography (not shown), sedimentation velocity ultracentrifugation,and electron microscopy (see below), which indicated an absenceof higher-order aggregates. However, at 4°C, and in theabsence of salt, ${sigma}$ A precipitates reversibly, indicating thatthe protein has a tendency to form higher-order aggregates.

In the crystal structure, 12 ${sigma}$ A molecules pack together intowhat at first view appears to be a double-helical assembly (Fig.4A). When the interactions are analyzed on the basis of buriedsurface area using the PISA server (21), three types of interactionswith potential biological relevance are observed. We have dividedthese possible interactions into three classes, the "tail-to-tail,""head-to-tail," and "head-to-head" interactions. The tail consistsof residues from the N-terminal loop, strand A, strand D, theloop to helix 4, the loop between strands G and H, and the loopbetween helix 8 and strand I, while the "head" consists of aminoacids from the loop between putative helix 2 and helix 3, fromhelix 10, and the loop between helices 10 and 11. The tail-to-tailinteraction has a buried surface of 690 Å², a solvationenergy gain of 12.4 kcal/mol, three hydrogen bonds, and a Pvalue of 0.033; the head-to-tail interaction has a buried surfaceof 467 Å², a solvation energy gain of 7.4 kcal/mol, sixhydrogen bonds, and a P value of 0.054, while the head-to-headinteraction buries a surface of 373 Å² and has a solvationenergy gain of 4.7 kcal/mol, two hydrogen bonds, and a calculatedP value of 0.26. None of the interactions observed among ${sigma}$ A moleculesin our crystal structure are similar to the previously observedinteractions among ${sigma}$ 2 molecules in the mammalian reovirus core.

The head-to-tail interaction is the only one involving all 12 ${sigma}$ A molecules of the asymmetric unit. It joins the 12 moleculesinto four trimers. Each trimer is related to its correspondingopposite in a double-helical hexamer by a rotation of about180°. The fit between trimers is very good (RMSD after superpositionof 0.4 Å), suggesting that the trimers act as a rigidbody. The monomers of the trimer are related by a rotation ofapproximately 50°, around an axis running through the centerof the double helix, which explains the previously reportedpeak in the self-rotation function at a chi angle of 50°(15). Interestingly, we note that canonical A-form dsRNA alsohas a helical rotation of around 50°.

The double-helical hexamer is related to the other hexamer inthe asymmetric unit by a rotation of around 180°. In thiscase, the fit between the two hexamers is not so good (RMSDafter superposition of 1.6 Å). It is tempting to proposethe trimer as a biological unit, arranged around dsRNA as asingle-stranded or double-stranded assembly. These models couldexplain the cooperative binding of ${sigma}$ A to dsRNA as well as thelarge complexes observed in the gel shift assays (Fig. 3B, middlepanel) (38). The previously discussed positively charged patchmaps to the inside of the helix (Fig. 4B). The width of thecavity inside the double-helical ${sigma}$ A assembly varies from 11 to35 Å, with the cross section being at least 20 Åwide in one dimension, while the diameter of A-form dsRNA isabout 10 by 20 Å in cross section. In other words, A-formdsRNA would probably fit, although some minor structural adjustmentsin the dsRNA or in the ${sigma}$ A assembly may be necessary.

The distance between Arg155 and Arg273 in the same moleculeis 8.5 Å, while the distances to their counterparts inthe next molecule of the trimer are 39 to 40 Å. In a modelconstructed with canonical A-form RNA and the crystallographictrimer, one arginine side chain could bind a phosphate on oneside of the minor groove and the other could bind a phosphateon the other side of the minor groove (distances between phosphates,10 Å). The next molecule could make the same interactionwith phosphates 13 bp further along the RNA chain (distancebetween phosphates, 38 Å) (Fig. 4B). A structural analysisof the ${sigma}$ A-dsRNA complex will be needed to confirm this hypothesis.Mutation of residues involved at the putative protein-proteininterface might also be attempted. However, inspection of thisinterface did not suggest any obvious target residues that,when mutated, would significantly affect protein-protein binding,without having a potential serious structural impact.

Stoichiometry of ${sigma}$ A binding to dsRNA. To obtain independent evidence for our dsRNA binding model andin an attempt to discriminate between our putative single anddouble ${sigma}$ A helices, we performed sedimentation velocity centrifugationexperiments on ${sigma}$ A-dsRNA complexes and transmission electron microscopyon negatively stained ${sigma}$ A-dsRNA samples.

In sedimentation velocity centrifugation experiments, a singlepeak was obtained in control experiments containing ${sigma}$ A or dsRNAalone, indicating that both are monodisperse. The estimatedmolecular masses were those expected for ${sigma}$ A monomers and dsRNAduplexes (Table 2). When excess protein was mixed with differentamounts of dsRNA (experiment 1), four reproducible peaks wereobtained. Sedimentation coefficients obtained for these peaksare compatible with monomeric ${sigma}$ A and three different complexesof ${sigma}$ A/dsRNA with stoichiometries of 1:1, 2:1, and 3:1 (Table2). In experiment 2 (Table 2), excess dsRNA was added to protein.Again, dsRNA of 36 bp gave a monodisperse peak, while in themixtures two or three peaks were observed, of which one clearlycorresponds to uncomplexed dsRNA. The complex peaks have smallersedimentation coefficients than expected for 1:1 and 2:1 ${sigma}$ A/dsRNAcomplexes, indicating that they are probably in equilibriumwith free dsRNA and 1:1 ${sigma}$ A/dsRNA complex, respectively. Takentogether, the centrifugation experiments suggest a maximum ofthree ${sigma}$ A molecules binding to a 36-bp dsRNA (Fig. 4B), whichwould be compatible with a single helix of ${sigma}$ A molecules coveringdsRNA.

View this table:
[in this window]
[in a new window]

TABLE 2. Sedimentation velocity centrifugation results and analysis of ${sigma}$ A/36-bp dsRNA mixtures

Electron micrographs (Fig. 5) also show multiple ${sigma}$ A moleculesbinding to dsRNA (Fig. 5C to F), while ${sigma}$ A alone appears monomeric(Fig. 5A and B). The width of the observed complexes variesfrom 54 to 98 Å and does not permit distinction betweena single helix (expected diameter, 60 Å) or double helices(expected diameter, 100 Å) of ${sigma}$ A binding to the dsRNA.In fact, binding may be plastic, with regions of dsRNA coveredby single-helical ${sigma}$ A and others covered by double-helical ${sigma}$ A,depending on the local dsRNA structure. Curiously, the structuresformed are clearly longer than the 36-bp dsRNA used, suggestingthat multiple dsRNA duplexes covered by ${sigma}$ A line up, presumablyvia ${sigma}$ A- ${sigma}$ A interactions. Future structural studies by cryo-electronmicroscopy and/or cocrystallization studies will hopefully shedmore light on the non-sequence-specific, cooperative mode ofdsRNA binding by RNA.

View larger version (107K):
[in this window]
[in a new window]

FIG. 5. Transmission electron microscopy of ${sigma}$ A-dsRNA mixtures. (A and B) ${sigma}$ A alone, which appears monomeric. Particles with approximate sizes of monomeric ${sigma}$ A are shown by circles. (C to F) Putative ${sigma}$ A-dsRNA complexes, indicated by arrows. Bar, 20 nm.

DISCUSSION

Top

DISCUSSION

Orthoreoviruses can be functionally classified into two subgroups,fusogenic and nonfusogenic, based on their ability to inducecell-cell fusion and form syncytia. The archetypal mammalianorthoreoviruses are nonfusogenic, although they do form specificpores in the membrane, proposed to be part of the membrane penetrationpathway during cell entry (1). The remaining members of thegenus, including avian orthoreoviruses (2), reptilian orthoreoviruses(10), baboon orthoreoviruses (8), and other mammalian orthoreoviruseslike Nelson Bay reovirus (9) and Pulau reovirus (32), are fusogenic.A series of diseases have been attributed to orthoreovirus infectionin animals, and recently it has been reported that Melaka reovirusof bat origin, another fusogenic mammalian reovirus, is associatedwith an acute respiratory disease in humans (5). However, infectionswith nonfusogenic orthoreoviruses are generally benign, withvery rare cases of mild upper respiratory tract illness or enteritisoccurring in infants and children.

Sequence analysis and RNA binding assays carried out on themajor outer capsid protein, ${sigma}$ B (and equivalents) from fusogenicreoviruses, have revealed that this protein, in contrast toits nonfusogenic counterpart ${sigma}$ 3, does not bind dsRNA (9, 37,38). As PKR downregulation in avian reovirus S1133 is causedby ${sigma}$ A (13), we analyzed the sequence of the minor inner capsidproteins of fusogenic and nonfusogenic reoviruses to see ifthe residues implicated in dsRNA binding are conserved amongthem. This analysis revealed that Arg273 is invariant amongfusogenic reoviruses (both avian and mammalian), whereas innonfusogenic reoviruses it is replaced by Thr. Arg155 is invariantamong avian reoviruses but is replaced by Gln in mammalian fusogenicreoviruses and by Gly in mammalian nonfusogenic reovirus. Thispattern of conservation suggests that the fusogenic counterpartsof the ${sigma}$ A protein retain their ability to bind dsRNA (and byextension the potential to downregulate PKR). In nonfusogenicreoviruses, the ${sigma}$ 2 proteins, which are structural homologuesof ${sigma}$ A, do not appear to bind dsRNA. Among this viral subclassRNA binding is linked to the outer capsid protein ${sigma}$ 3. These differencesin function may profoundly influence host cell interactionsand could be related to and/or have evolved together with thesyncytial mode of propagation of the fusogenic viruses and arethus intimately tied to their pathogenicity.

In conclusion, the structure, gel shift, sedimentation velocity,and electron microscopy data presented here suggest a cooperativemode of ${sigma}$ A binding to dsRNA, which may be of general relevanceto all fusogenic reoviruses, thereby contributing to a betterunderstanding of their biology. This improved understandingmay in turn lead to better strategies to combat these viruses.

ACKNOWLEDGMENTS

This research was sponsored by research grants BFU2005-02974and BFU2005-24982-E (awarded to M.J.V.) and by a predoctoralFPU fellowship to P.G.-C., all from the Spanish Ministry ofEducation and Science, and by research grant PGIDIT04BTF203003PRfrom the Xunta de Galicia (awarded to J.M.-C.). As part of theEuropean Science Foundation EUROCORES Programme EuroSCOPE, thework was also supported by funds from the European Commissionunder contract ERAS-CT-2003-980409.

We thank Carlos Alfonso and Germán Rivas of the CSIC(Centro de Investigaciones Biológicas, Madrid, Spain)for help with the analytical ultracentrifugation experimentsand Patricia Ferraces Casais and Rebeca Menaya Vargas for technicalassistance.

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Bioquímica e Bioloxía Molecular, Facultade de Farmacia, Universidade de Santiago de Compostela, E-15782 Santiago de Compostela, Spain. Phone and fax: 34 981 599157. E-mail: mark.vanraaij{at}usc.es

Published ahead of print on 17 September 2008.

REFERENCES

Top