Structure of deformed wing virus, a major honey bee pathogen

Karel Škubník; Jiří Nováček; Tibor Füzik; Antonín Přidal; Robert J Paxton; Pavel Plevka

doi:10.1073/pnas.1615695114

. 2017 Mar 7;114(12):3210–3215. doi: 10.1073/pnas.1615695114

Structure of deformed wing virus, a major honey bee pathogen

Karel Škubník ^a, Jiří Nováček ^a, Tibor Füzik ^a, Antonín Přidal ^b, Robert J Paxton ^c, Pavel Plevka ^a,¹

PMCID: PMC5373406 PMID: 28270616

Significance

Honey bee populations in Europe and North America have been decreasing since the 1950s. Deformed wing virus (DWV), which is undergoing a worldwide epidemic, causes the deaths of individual honey bees and collapse of whole colonies. We determined three-dimensional structures of DWV at different conditions and show that the virus surface is decorated with protruding globular extensions of capsid proteins. The protruding domains contain a putative catalytic site that is probably required for the entry of the virus into the host cell. In addition, parts of the DWV RNA genome interact with the inside of the virus capsid. Identifying the RNA binding and catalytic sites within the DWV virion offers prospects for the development of antiviral treatments.

Keywords: colony collapse disorder, virus, structure, Apis mellifera, honey bee

Abstract

The worldwide population of western honey bees (Apis mellifera) is under pressure from habitat loss, environmental stress, and pathogens, particularly viruses that cause lethal epidemics. Deformed wing virus (DWV) from the family Iflaviridae, together with its vector, the mite Varroa destructor, is likely the major threat to the world’s honey bees. However, lack of knowledge of the atomic structures of iflaviruses has hindered the development of effective treatments against them. Here, we present the virion structures of DWV determined to a resolution of 3.1 Å using cryo-electron microscopy and 3.8 Å by X-ray crystallography. The C-terminal extension of capsid protein VP3 folds into a globular protruding (P) domain, exposed on the virion surface. The P domain contains an Asp-His-Ser catalytic triad that is, together with five residues that are spatially close, conserved among iflaviruses. These residues may participate in receptor binding or provide the protease, lipase, or esterase activity required for entry of the virus into a host cell. Furthermore, nucleotides of the DWV RNA genome interact with VP3 subunits. The capsid protein residues involved in the RNA binding are conserved among honey bee iflaviruses, suggesting a putative role of the genome in stabilizing the virion or facilitating capsid assembly. Identifying the RNA-binding and putative catalytic sites within the DWV virion structure enables future analyses of how DWV and other iflaviruses infect insect cells and also opens up possibilities for the development of antiviral treatments.

The western honey bee (Apis mellifera) plays a vital role in world agriculture by providing pollination services to diverse commercial crops, a service valued at US$ 215 billion annually (1). In addition, honey bees pollinate numerous wild flowering plants, thereby supporting biodiversity (2, 3). However, over the past two decades, honey bees have suffered from elevated mortality in North America and Europe (4, 5). Colony losses have been associated with the exotic ectoparasitic mite Varroa destructor, which feeds on honey bee hemolymph, thereby vectoring numerous honey bee viral pathogens, in particular the iflavirus deformed wing virus (DWV). In the absence of varroa, DWV levels are low, and the virus causes asymptomatic infections. Varroa-infested colonies show elevated levels of DWV (6, 7). Symptoms associated with acute DWV infections include the death of pupae, as well as deformed wings, shortened abdomen, and cuticle discoloration of adult bees that die soon after pupation, causing colony collapse (6, 8). Indeed, winter colony mortality is strongly correlated with the presence of DWV, irrespective of the levels of varroa infestation (8, 9). DWV-induced loss of honey bees, coupled with a long-term decline in beekeeping, has become a serious threat to adequate provision of pollination services, threatening food security and ecosystem stability (1).

Viruses from the order Picornavirales, including the family Iflaviridae, have nonenveloped icosahedral virions that are about 30 nm in diameter (10). Iflavirus capsids protect 10,000-nt-long ssRNA genomes, which are translated into polyproteins that are cotranslationally and posttranslationally cleaved by viral proteases to produce structural (capsid-forming) and nonstructural proteins (11). The major capsid proteins VP1, VP2, and VP3 originating from a single polyprotein form a protomer, the basic building block of the pseudo-T3 icosahedral capsid. The entire capsid consists of 60 such protomers, arranged in 12 pentamer units of 5 protomers each.

Previously, the structure of the iflavirus Chinese sacbrood virus was characterized to a resolution of 25 Å by cryo-electron microscopy. The structure confirmed the pseudo-T3 icosahedral symmetry of its capsid and a smooth outer surface of the virion (12). Recently, we determined the structure of the iflavirus slow bee paralysis virus (SBPV) to a resolution of 2.6 Å by X-ray crystallography (13). Despite its efficient transmission by V. destructor, SBPV infection is a rare disease of honey bees (14). The structure revealed that the C-terminal extension of capsid protein VP3 of SBPV forms a globular protruding (P) domain positioned at the virion surface. The P domain is anchored to the core of the VP3 subunit by a 23-residue-long flexible linker that allows the P domain to attach to different areas of the capsid (13). In addition, the P domain contains the putative active site Asp-His-Ser, which is conserved among several iflaviruses (13). Iflaviruses were also proposed to harbor short VP4 subunits consisting of only about 20 residues (11, 14); however, electron density corresponding to SBPV VP4 was not identified in the SBPV virion structure (13).

Here, we present the structure of the DWV virion and show that, similar to SBPV, it contains a C-terminal extension of capsid protein VP3 that forms a globular domain with a putative receptor-binding site located at the virion surface. We show that, unlike SBPV, DWV’s putative active site not only is flexible but also adopts two alternative conformations. Furthermore, bases from the RNA genome interact with the DWV capsid close to the fivefold axes. These structural details provide potential targets for development of antiviral compounds.

Results and Discussion

Structure of DWV Virion and Capsid Proteins.

The structure of the DWV virion was determined to a resolution of 3.5 Å using cryo-electron microscopy and to 3.8 Å using X-ray crystallography (Table S1 and Fig. S1). The DWV virion is built from subunits VP1, VP2, and VP3 arranged in a capsid with pseudo-T3 icosahedral symmetry (Fig. 1). The major capsid proteins have jellyroll β-sandwich folds with β-strands named according to the virus jellyroll fold convention B to I (Fig. 1A) (15, 16). The complete structures of the major capsid proteins could be built except for residues 1 and 254 to 258 out of the 258 residues of VP1, 251 to 254 out of the 254 residues of VP2, and 1 and 416 out of the 416 residues of VP3. Iflaviruses were suggested to harbor short VP4 subunits consisting of about 20 residues (11, 14). Nevertheless, the electron density corresponding to VP4 could not be identified in either of the two DWV virion structures.

Table S1.

DWV structure quality indicators

Parameter	Cryo-EM structure of native DWV virion	Cryo-EM structure of DWV virion incubated in crystallization buffer and cross-linked with 1% glutaraldehyde	Cryo-EM structure of DWV virion incubated in crystallization buffer	Cryo-EM structure of DWV virion at pH 5.0	Crystal structure of DWV virion	Crystal structure of P domain
EMDB accession no.	EMD-4014	EMD-3574	EMD-3570	EMD-3575	NA	NA
PDB ID code	5L8Q	5MV5	5MUP	5MV6	5G52	5G51
Space group	NA	NA	NA	NA	I23	C222
a, b, c, Å	NA	NA	NA	NA	360.13, 360.13, 360.13	63.97, 104.92, 57.95
α, β, γ, °	NA	NA	NA	NA	90, 90, 90	90, 90, 90
Resolution, Å	3.5	3.1	3.8	4.1	29.8–3.8 (3.9–3.8)^*	39.8–1.45 (1.47–1.45)^*
R_merge^†	NA	NA	NA	NA	0.37 (0.81)^*	0.06 (0.54)^*
〈I〉/〈σI〉	NA	NA	NA	NA	2.0 (0.9)^*	14.2 (2.1)^*
Completeness, %	NA	NA	NA	NA	75.7 (76.1)^*	99.9 (98.4)^*
Redundancy	NA	NA	NA	NA	1.8	6.1
No. of reflections	NA	NA	NA	NA	102,129	212,754
R_work/R_free	0.296/0.336	0.310/0.312	0.362/0.379	0.337/0.381	0.301/0.337	0.189/0.212
No. of atoms	7,111^‡	7,005^‡	6,673^‡	6,857^‡	7,047^‡	1,457 protein, 242 water
rmsd bond lengths, Å	0.01	0.013	0.010	0.009	0.006	0.006
rmsd bond angles, °	1.151	1.376	1.318	1.324	1.284	1.047
Ramachandran favored, %^§	90.9.	92.4	92.9	92.4	81.6	97.2
Ramachandran allowed, %^§	8.3	7.6	7.1	7.6	16.2	2.8
Ramachandran outliers, %^§	0.8	0	0	0	2.2	0
Poor rotamers, %^§	0	0.5	0.6	0.1	8.1	2
Clashscore (percentile)	15.99 (95)	7.83 (82)	9.82 (72)	13.16 (57)	23.07 (89)	7.05 (80)
MolProbity score (percentile)	2.23 (99)	1.90 (81)	1.97 (77)	2.10 (70)	3.24 (81)	1.76 (63)
Cβ deviations, %^§	0	0	0	0	0.05	0
CC^¶	NA	NA	NA	NA	0.807 (0.618)	NA
Average B-factor of protein atoms, Å²	122.8	85.8	110.3	149.1	45.7	24.2

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NA, not applicable.

Statistics for the highest resolution shell are shown in parentheses.

^{^†}

R_merge = Σ_hΣ_j|l_hj − 〈l_h〉|/ΣΣ|l_hj|.

^{^‡}

Statistics are given for one icosahedral asymmetric unit.

^{^§}

According to the criterion of Molprobity (46).

^{^¶}

Correlation coefficient of observed structure factor amplitudes and structure factor amplitudes calculated from NCS-averaged map.

Fig. S1. — FSC curves of cryo-EM reconstructions of DWV virions in different conditions. The FSC of the native DWV virion is shown in red, DWV at pH 5.0 in blue, DWV exposed to the crystallization condition in green, and DWV exposed to the crystallization condition and cross-linked by glutaraldehyde in yellow.

Subunit VP3 of DWV contains a C-terminal extension that forms a P domain positioned on the virion surface (Fig. 1). Because of the P domains, the maximum diameter of the DWV virion (397 Å) is similar to that of SBPV (398 Å) and bigger than those of other picornaviruses and dicistroviruses (about 300 Å). The cryo-EM and X-ray structures of the capsid shell of DWV are similar, with an rmsd of Cα-atoms of 0.77 Å; however, the positioning of the P domains on the surface of the virions is different (Fig. 1 B and C). The location of the P domains in the crystal structure is not affected by crystal contacts, indicating that it depends on the composition of the solution surrounding the virus. The cryo-EM images were collected on virions in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) whereas the crystallization conditions contained 0.8 M KH₂PO₄, 0.8 M NaH₂PO₄, and 0.1 M sodium Hepes, pH 7.5. The movement of the P domain between the two alternative attachment sites at the virion surface involves a 39-Å shift of its center of mass and 145° rotation. This change in position is possible due to a 20-residue-long linker that connects the P domain to the core of the VP3 subunit.

Host cell entry of iflaviruses has not been studied, but it probably includes receptor-mediated endocytosis, as has been demonstrated for some picornaviruses (17, 18). Endosomal entry involves exposure of the virions to a solution differing in composition (e.g., low pH), which could trigger detachment of the P domain from the virion surface. It is possible that, during cell entry, the P domain functions without being fixed to a specific position at the virion surface. Instead, it may be anchored by its polypeptide linker to the core of the VP3 subunit. Similar, alternative attachment sites of the P domain at the virion surface have been observed previously for SBPV, reinforcing the possibility that movement of the P domains might be required for their biological function (13). It was speculated previously that the movements of the SBPV P domains were induced by differences in pH. In contrast, the DWV structures were determined at similar pH; however, the crystallization solution was of high ionic strength (0.8 M KH₂PO₄, 0.8 M NaH₂PO₄). It is therefore possible that the movements of the P domain of DWV that we observed were induced by these high, nonphysiological ion concentrations.

Structure of the P Domain.

The P domain of the VP3 subunit of DWV is globular, with a diameter of 30 Å (Fig. 1). In both cryo-EM and X-ray structures of the DWV virion, the residues of the P domain have higher average temperature factors than residues from the remainder of the capsid, indicating a higher mobility of the P domain. As a result, electron density maps of the P domains are less well-resolved than other parts of the capsid. We therefore used X-ray crystallography to determine the structure of the isolated P domain to a resolution of 1.45 Å (Table S1). In addition to the globular core, the P domain of DWV contains a finger-shaped loop with four-residue-long antiparallel β-strands β8 and β9 (Fig. 2A). The core of the P domain consists of a central antiparallel β-sheet formed from strands β5 and β6 surrounded by the 11-residue-long α-helix α1, the 5-residue long α2, and two β-sheets containing strands β1 and β2 and β3 to β7 (Fig. 2A). The β-strands are connected by loops that vary in length between 6 and 45 residues. The high-resolution structure of the P domain was fitted into the corresponding regions of the cryo-EM and crystallographic electron density maps of the DWV virion and refined against the experimental data. The resulting P domain structures are similar, with an rmsd in atom positions smaller than 0.50 Å. However, residues 399 to 416 forming the P domain “finger” were not resolved in either of the virion electron density maps, probably due to the flexibility of the loop.

Fig. 2. — Structure of the DWV P domain, its localization on the virion surface, and details of the putative catalytic or receptor-binding site. Schematic representation of the crystal structure of the P domain (A), rainbow-colored from residue 260 in blue to 416 in red. Atoms of residues forming the putative active site Asp294, His277, and Ser278 are shown as spheres. Please note that β-strand 4, which is present in the P domain of SBPV, is missing in the DWV structure. Surface representation of pentamers of capsid protein protomers of (B) native cryo-EM structure and (C) X-ray structure of DWV virions. VP1 subunits are shown in blue, VP2 in green, VP3 in red, and the P domain in magenta. Positions of the conserved residues that form a putative active or receptor-binding site of DWV are highlighted in cyan. The borders of one icosahedral asymmetric unit are highlighted with a black triangle. (*Insets*) Detailed views of the conserved residues forming a compact patch at the P domain surface. In the *Inset* of B, one of the P domains was removed to allow a view of the inside of the crown formed by the P domains. The P domains of DWV (D) and SBPV (E) contain putative Ser-His-Asp active sites, which are parts of groups of seven residues that are conserved among iflaviruses. The residues are displayed in stick representation. The conserved residues are shown with carbon atoms in cyan. Residues that are not conserved are shown with carbon atoms in magenta. Residues Ser278, Ala292, Ser293, and Asp294 of DWV adopt alternative conformations that are shown with carbon atoms in yellow. Distances between the side chains constituting the putative catalytic triad are shown as orange dashed lines. The electron density maps are contoured at 1 σ.

Comparison of DWV and SBPV Virion Structures.

DWV shares 32% sequence identity in capsid proteins with SBPV (13). The two viruses have similar surface topologies with capsids decorated with P domains. The differences between the SBPV and DWV capsids are predominantly in the loops of the major capsid proteins exposed at the outer virion surface. Capsid protein VP1 of DWV has a GH loop that is five residues shorter than that of SBPV and lacks an α-helix α6 (Fig. S2 A and B). The GH loop of the VP2 subunit of SBPV contains the integrin-recognition motif Arg192-Gly193-Asp194 (RGD) and is four residues longer than that of DWV (Fig. S2 C and D). Integrins serve as cell entry receptors for numerous viruses, including the foot and mouth disease virus and several parechoviruses (19–21). However, the RGD motif is not present in DWV and other iflaviruses. Differences in the structure of the GH loop between DWV and SBPV might reflect different functions of the loops in receptor recognition.

DWV and SBPV differ in the structures of both the core and P domains of their VP3 subunits (Fig. S2 E and F). The β-sandwich cores of VP3 subunits of DWV and SBPV can be superimposed with an rmsd of 1.35 Å for 94% of the residues whereas the P domains have an rmsd of 1.69 Å for 80% of the residues. The P domain of DWV contains 18 structured residues at the C terminus that form a finger with a 4-residue-long antiparallel beta sheet (Fig. 2A and Fig. S2G). Notably, these residues are not resolved in the crystal structures of SBPV virions (Fig. S2H). The sequence identity within the P domains of the two viruses is 17% whereas it is 33% for the remaining parts of the capsid proteins. Thus, it seems that the P domain is more tolerant to mutations than the parts of the virus proteins forming the capsid shell. Nevertheless, the P domains of DWV and SBPV contain 8 conserved residues that are also shared with several other iflaviruses and that might form a catalytic or receptor-binding site as discussed below.

Comparison of the P Domain with Other Proteins.

A search for structures similar to the DWV P domain (22) identified a globular surface domain of a virus from the Astroviridae, an additional family of nonenveloped viruses like the picornaviruses and caliciviruses, all of which possess a single-stranded positive sense RNA genome (Table S2) (23). The domain is similar to the P domain of DWV in terms of having a core formed by β-strands that are complemented by short α-helices located at the periphery of the domain. Nevertheless, the two domains are quite different and could not be meaningfully superimposed. Several additional proteins could be detected, but their structural similarities to the DWV P domain were low and the alignments always included only a small fraction of the structures (Table S2). Therefore, it seems that the P domains of iflaviruses might have evolved de novo as C-terminal extensions of the capsid proteins and therefore have a unique fold.

Table S2.

Proteins identified based on structural similarity to P domain of DWV

Protein	PDB ID code	Organism	Z-score	rmsd	Fraction of aligned residues, %	Sequence identity, %
SBPV VP3	5j98	SBPV	17.1	2.1	83	17
Human astrovirus capsid protein	5IBV	Mamastrovirus 1	4.7	3.3	57	4
Focal adhesion kinase	2J0K	Gallus gallus	2.9	3.8	37	6
Carbonic anhydrase II	3M14	Homo sapiens	2.9	3.6	38	2
l-fuconate dehydratase rTSγ	4A35	Homo sapiens	2.1	3.6	41	3
Glycyl-tRNA synthetase	4KQE	Homo sapiens	2.0	3.8	37	8

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Position of the P Domain at the Virion Surface.

In the cryo-EM structure of DWV, the P domains related by one icosahedral fivefold axis form a crown-like arrangement at the virion surface (Figs. 1B and 2B). The crowns have a diameter of 80 Å and protrude 40 Å above the capsid surface. The P domains within the same crown contact each other with a buried surface area of 380 Å², and each of them binds to the capsid through a 430-Å² interface located next to the fivefold axis. In contrast, the P domains of native SBPV, which also forms crowns, are not in contact with each other (Fig. S3). In the crystal structure of DWV, the P domains are positioned approximately in between the icosahedral fivefold, threefold, and twofold axes and interact with the capsid through a 1,000-Å² interface (Fig. 2C). The P domains in this structure do not interact with each other, and, therefore, we refer to them as being in “centered” arrangement. The movements of the P domains between the two positions seem to be accomplished by “rolling” over the virion surface (Fig. 2 B and C and Movies S1 and S2). The residues forming the 430 Å² of the VP1 surface that became exposed after the P domain movements do not contain any specific motives to indicate their function in receptor binding. Similar movements of the P domains have been observed previously in SBPV crystallized in low pH conditions (13). Unlike in the case of SPBV, movements of the DWV P domain could not be induced by exposing DWV to pH 5.0 (Fig. S4 A and B). However, the structures of the P domains determined by cryo-EM of DWV at low pH are less resolved and therefore more mobile than those of the virus in neutral pH (Fig. S5 A and B). To determine whether the movements of the P domains are reversible, we exposed DWV virions to the crystallization condition and subsequently dialyzed them back against PBS. Cryo-EM reconstruction of these particles determined to a resolution of 3.8 Å showed P domains in the crown arrangement similar to native virions (Figs. S4C and S5C). It was not possible to calculate cryo-EM reconstruction of the DWV virions in the crystallization buffer because the 1.6-M phosphate salts prevent preparation of grids with vitreous ice of sufficient quality for cryo-EM data collection. In an attempt to directly observe the virus with P domains in the centered arrangement, DWV virions were exposed to the crystallization condition and cross-linked by addition of 1% glutaraldehyde. The particles were then dialyzed against PBS and used for cryo-EM reconstruction that was determined to a resolution of 3.1 Å. These particles had P domains in the crown arrangement (Figs. S4D and S5D). Three-dimensional classification using the program RELION did not identify a subclass of particles with P domains in the centered arrangement. Thus, the cryo-EM analysis did not confirm the positioning of P domains in the centered arrangement observed in the crystal structure (Fig. 1C). The differences in the virion structures obtained by the two types of structural analysis could be caused by low-efficiency of cross-linking of the P domains. Furthermore, it is likely that the movements of P domains within a single virus particle are not synchronized with each other. Cryo-EM analysis, even in combination with 3D classification, may therefore not allow detection of a subset of the P domains in the centered arrangement. In contrast, crystallization, which required 5 months, probably specifically selected for particles with a centered P domain arrangement.

Fig. S3. — Comparison of positioning of P domains at the virion surfaces of native DWV and SBPV. Surface representation of pentamers of capsid protein protomers of DWV (A) and SBPV (B). VP1 subunits are shown in blue, VP2 in green, VP3 in red, and the P domains in magenta. The conserved residues that form putative active or receptor-binding sites are highlighted in cyan. The borders of one icosahedral asymmetric unit are highlighted with a black triangle.

Fig. S4. — Molecular surfaces of DWV virions at different conditions determined by cryo-EM. Native DWV (A), DWV at pH 5.0 (B), DWV exposed to the crystallization condition (C), and DWV exposed to the crystallization condition and cross-linked (D). The virion surfaces are rainbow-colored according to their distance from the particle center.

Fig. S5. — Cryo-EM reconstructions of DWV virions in different conditions shown as molecular surfaces colored according to local resolutions of the structures. Individual scale bars for the local resolutions are shown below each reconstruction. Native DWV (A), DWV at pH 5.0 (B), DWV exposed to the crystallization condition (C), and DWV exposed to the crystallization condition and cross-linked (D).

P domains of DWV virions that were exposed to high salt or low pH became more mobile than those of native virions or viruses cross-linked by 1% glutaraldehyde (Fig. S5). We speculate that it is the mobility of the P domains rather than their precise positioning that is important for DWV cell entry, during which the virus is likely to encounter low pH. Our results provide evidence of the possible extent of movements of the P domains.

Putative Role of the P Domain in Cell Entry.

The P domain of DWV contains residues Asp294, His277, and Ser278 located close to each other, and the arrangement of their side chains indicates that they constitute a catalytic triad (Fig. 2 A and D) (24). The distances between the side chains of the residues are larger than ideal for catalyzing a hydrolytic reaction (Fig. 2D) (24). However, the 1.45-Å-resolution structure of the DWV P domain shows that residues Ser278, Ala292, Ser293, and Asp294 adopt alternative conformations (Fig. 2D), indicating local flexibility of the structure. It is therefore possible that the optimal configuration of the active site might be achieved upon binding the as-yet-unknown substrate. This type of catalytic triad has been previously identified in proteases, lipases, and esterases (24–26). Therefore, DWV may use the putative catalytic activity of its P domains in cell entry. The P domains might bind to virus receptors or disrupt membranes and thus allow the virus to deliver its genome into the cell cytoplasm.

The putative catalytic triad and five additional residues, which form a compact patch on the P domain surface, are conserved among iflaviruses containing P domains (Figs. 2 B and C and 3A) (11, 27–29), which is in contrast to the limited 3% overall sequence identity of the remaining residues of the P domains. Conservation of these residues indicates that they constitute a functionally important receptor-binding or substrate-binding site. The Asp294-His277-Ser278 catalytic triad of SBPV also has a 3D arrangement indicative of an active site (Fig. 2E) (13). A similar group of residues in the P domains of noroviruses was shown to bind glycans (30, 31). Alternatively, the putative catalytic site may facilitate the escape of DWV virions from endosomes in a manner analogous to the lipase activity present in the N-terminal domain of the capsid protein VP1 from parvoviruses (32).

Fig. 3. — Sequence alignment of residues of iflavirus VP3 subunits. API, *Antherae pernyi* iflavirus; HEI, *Heliconius erato* iflavirus. UniProt accession numbers of the sequences used in the alignment are provided. (A) Conserved residues constituting the putative catalytic triad Asp-His-Ser are highlighted with an orange background; other conserved residues located in the structure close to the putative active site are highlighted with a gray background. (B) Conserved residues involved in the interaction with genomic RNA are highlighted with a yellow background.

The conserved residues in the iflavirus P domain provide a potential target for antiviral compounds. The putative active site faces the interior of the crown in the native virus; however, it is exposed at the apex of the P domain in the virion structure with the centered arrangement of the P domains (Fig. 2 B and C). The exposure of the active site after the P domain rotation reinforces the possibility that movements of the P domain might be required for efficient DWV infection.

Evolutionary Relationship to Other Viruses from the Order Picornavirales.

The availability of the DWV and SBPV virion structures enabled the construction of a structure-based phylogenetic tree comparing the iflaviruses to other viruses from the families Dicistroviridae and Iflaviridae (Fig. 4). The structural comparison shows that iflaviruses are most similar to the insect-infecting dicistroviruses Israeli acute bee paralysis virus, cricket paralysis virus, and triatoma virus (33–35). The viruses most similar to DWV and SBPV from the Picornaviridae family are hepatitis A virus and human parechovirus 1 (HPeV-1), which were previously suggested to form evolutionary intermediates between human and insect viruses (21, 36) (Fig. 4). The closer structural similarity of DWV and SBPV capsid to the viruses from the Dicistroviridae family than to viruses from the Picornaviridae family might be because of similarities in the processing of the polyprotein precursor of capsid proteins. The amino acid sequence of the VP4 subunit is located in front of the N terminus of VP3 in viruses from the family Dicistroviridae whereas it is located in front of the VP2 sequence in viruses of the family Picornaviridae. The VP4 sequences of iflaviruses were predicted to be located in front of VP3 in the polyprotein (11, 14). Even though the VP4 subunits of DWV and SBPV are not resolved in the virion structures, the similar cleavage pattern of the capsid protein subunits from the precursor polyprotein P1 might impose constraints on the organization of the capsid, resulting in the closer similarity of iflaviruses to dicistroviruses.

Fig. 4. — Evolutionary relationship among viruses from the families *Iflaviridae*, *Picornaviridae*, and *Dicistroviridae* based on structural alignment of their capsid proteins. Phylogenetic tree based on structural similarity of icosahedral asymmetric units of indicated viruses. For details on the construction of the diagram, please *Materials and Methods*.

Iflaviruses are structurally and genetically related to vertebrate picornaviruses, for which numerous capsid-binding inhibitors have been developed. Compounds that bind into a hydrophobic pocket within VP1 can inhibit receptor binding and/or genome release of some picornaviruses (37–39). However, such a hydrophobic pocket is not formed in DWV VP1 subunits. Similarly, the hydrophobic pocket was not observed in VP1 of SBPV (13), which suggests that capsid binding inhibitors that intercalate into VP1 subunits may not be effective as antivirals against honey bee viruses of the family Iflaviridae.

Capsid–RNA Interactions.

The DWV genome is a 10,140-nt-long linear RNA molecule (11) that forms unique interactions with the inner surface of the icosahedral capsid. The virus RNA does not affect the packing of particles within the crystal or the determination of particle orientations performed in the course of the cryo-EM reconstruction. Therefore, both X-ray and cryo-EM maps contain information about the icosahedrally averaged RNA structure.

Clearly defined electron density corresponding to an RNA nucleotide is associated with each VP3 subunit of DWV close to the fivefold icosahedral axis (Fig. 5A). The shape of the density indicates that the base of the nucleotide is a pyrimidine, and it was therefore modeled as a uridine (Fig. 5B). The nucleotide has 90% occupancy, showing that the genome binds to nearly all of the 60 available positions within the virion. Each nucleotide interacts with residues from three VP3 subunits belonging to different protomers within one pentamer (Fig. 5B). The residues that bind the RNA are conserved among several honey bee Iflaviruses (Fig. 3B). However, the structured RNA was not observed in the SBPV virion, which does not have the conserved RNA-binding residues (Fig. 3B) (40). Reminiscent of the RNA–protein interactions in DWV are structured RNA oligonucleotides that have been recently described in the parechoviruses HPeV-1, HPeV-3, and Ljungan virus, where they also mediate contacts among capsid proteins from different protomers (21, 41, 42). The conservation of the RNA-binding residues among some of the honey bee iflaviruses, together with the near-complete occupancy of the RNA, indicates that the RNA–capsid binding might play a role in virion stability. In addition, RNA–capsid interactions may play a role in DWV virion assembly, as was previously suggested for related picornaviruses (43). Therefore, future mutational analyses of the residues involved in the RNA binding may lead to determination of the mechanism that ensures packaging of the DWV genome into newly forming particles, and which may offer alternative targets for antiviral compounds.

Fig. 5. — Interactions of DWV genomic RNA with capsid. Location of RNA nucleotides displayed in yellow within the pentamer of capsid protein protomers as seen from the inside of the virion (A). VP1 subunits are shown in blue, VP2 in green, and VP3 in red. The borders of one icosahedral asymmetric unit are highlighted with a triangle. (B) Detail of the interaction of viral RNA with the VP3 subunits. The electron density map of the nucleotide is contoured at 1 σ. VP3 subunits from different icosahedral asymmetric units are distinguished by color shades and superscripts “A,” “B,” and “C.”

Materials and Methods

The propagation of DWV was carried out as described in the COLOSS BeeBook (44). A suspension of DWV was applied onto holey carbon grids and vitrified by plunging into liquid ethane. Images were recorded with an FEI Falcon II camera in an FEI Titan Krios electron microscope. The images were processed using the package RELION (45). The P domain was expressed in Escherichia coli BL21(DE3). Crystals of the DWV P domain were obtained using the sitting-drop technique with a bottom solution containing 4.3 M sodium chloride and 0.1 M Hepes, pH 7.5.

SI Materials and Methods

Virus Propagation in Honey Bee Pupae.

The propagation of DWV was carried out as described in the COLOSS BeeBook (44). Brood areas with Apis mellifera white-eyed pupae were identified by color and the structural features of the cell caps. White-eyed pupae were carefully extracted from the brood combs so as not to injure the pupae. The pupae were placed on paper furrows with their ventral side up. In total, 2,581 pupae were used for DWV propagation. The virus inoculum (1 μL) was injected into pupae using a Hamilton micropipettor with a 30-gauge, 22-mm-long needle through the intersegmental cuticle between the fourth and fifth sternite. Pupae that leaked hemolymph after the injection were discarded. The optimal concentration of the virus in the inoculum was determined empirically by comparing virus yields when using different virus concentrations in the inoculum. Inoculated pupae were placed into Petri dishes with the paper furrows and incubated at 30 °C and 75% relative humidity for 5 d. After incubation, the pupae were frozen at −20 °C. For long-term storage, the pupae were kept at −80 °C.

Virus Purification.

Fifty to seventy experimentally infected honey bee pupae were homogenized with a Dounce homogenizer (piston-wall distance 0.075 mm) in 30 mL of PBS, pH 7.5 (Sigma-Aldrich). The extract was centrifuged at 15,000 × g for 30 min at 10 °C. The pellet was discarded, and the supernatant was ultracentrifuged at 150,000 × g for 3 h in a Ti50.2 fixed angle rotor (Beckman-Coulter). The resulting pellet was resuspended in PBS to a final volume of 5 mL. MgCl₂ was added to a final concentration of 5 mM as well as 20 μg/mL DNase I and 20 μg/mL RNase. The solution was incubated at room temperature for 30 min and centrifuged at 5,500 × g for 15 min. The resulting supernatant was separated on a CsCl (0.6 g/mL) gradient in PBS by ultracentrifugation for 16 h at 30,000 rpm in an SW40 swinging bucket rotor (Beckman-Coulter). Virus bands were collected by the gentle piercing of ultracentrifuge tubes with an 18-gauge needle. The viruses were buffer-exchanged to PBS and concentrated using centrifuge filter units with a 100-kDa molecular mass cutoff. This procedure yielded about 300 μg of virus with purity sufficient for cryo-EM and crystallization.

Single Particle Data Acquisition and Image Processing.

The following samples were used for cryo-EM analyses: (i) freshly purified DWV in PBS; (ii) DWV incubated in the crystallization solution containing 0.8 M potassium dihydrogen phosphate, 0.8 M sodium dihydrogen phosphate, 0.1 M sodium Hepes, pH 7.5 for 12 h; subsequently, the virus was dialyzed against PBS; (iii) DWV incubated in the crystallization solution for 12 h and then cross-linked by 1% glutaraldehyde for 1 h; subsequently, the virus was dialyzed against PBS; and (iv) DWV incubated in phosphate buffer with pH 5.0 at 34 °C for 20 min. A suspension of DWV (3.5 μL at concentration 2 mg/mL) was applied onto holey carbon grids (Quantifoil R2/1, mesh 300; Quantifoil Micro Tools) and vitrified by plunging into liquid ethane using an FEI Vitrobot Mark IV. Grids with the vitrified sample were transferred to an FEI Titan Krios electron microscope operated at 300 kV aligned for parallel illumination in nanoprobe mode. The sample in the column of the microscope was kept at −196 °C. Images were recorded with an FEI Falcon II direct electron detection camera under low-dose conditions (20 e⁻/Å²) with underfocus values ranging from 1.0 to 2.9 µm at a nominal magnification of 75,000, resulting in a pixel size of 1.07 Å/pixel. Each image was recorded in movie mode with 0.38 s total acquisition time and saved as seven separate movie frames. The frames from each exposure were aligned to compensate for drift and beam-induced motion during image acquisition using the program SPIDER (47).

Icosahedral Reconstruction of the DWV Virion.

Regions of the images containing the DWV virions (512 × 512 pixels) were extracted from the micrographs using the program e2boxer.py from the package EMAN2 (48), resulting in 136,834 particles. Contrast transfer function (CTF) parameters of incoherently averaged particles from each micrograph were automatically estimated using the program ctffind3 (49). Subsequently, the particles were separated into two half-datasets for all of the subsequent reconstruction steps to follow the “gold standard” procedure for resolution determination (50). A 7.5-Å-resolution model of DWV determined by X-ray crystallography was used to initiate the reconstruction. The phases of the initial model were randomized beyond a resolution of 30 Å. The images were processed using the package RELION (45). The dataset was subjected to multiple rounds of 2D classification and 3D classification, resulting in a near-homogeneous set of full DWV particles. Subsequent refinement was performed using the 3dautorefine procedure, with the starting model from the previous reconstruction low pass-filtered to a resolution of 60 Å. The reconstruction was followed by another round of 3D classification, where the alignment step was omitted and the estimated orientations and particle center positions from the previous refinement step were used. The final reconstruction was performed using the program 3dautorefine. The resulting unfiltered electron density maps were masked by threshold masks created using the RELION mask_create function. To avoid overmasking, the masked maps were visually inspected to exclude the possibility of clipping of electron densities belonging to the virus capsids. Additionally, the occurrence of overmasking was monitored by Fourier shell correlation (FSC) curve shape inspection. Furthermore, the shapes of the FSC curves of phase-randomized half-datasets with the applied mask were checked (51). The resulting resolutions of the reconstructions were estimated as the values at which the FSC curves fell to 0.143.

Cryo-EM Structure Determination and Refinement.

The initial model, derived from the CrPV structure (34), was fitted into the B-factor–sharpened cryo-EM map of DWV and subjected to manual rebuilding using the program Coot and coordinate and B-factor refinement using the program package Phenix (52, 53).

Crystallization of the DWV Virion.

DWV crystallization screening was performed at 20 °C using the virus in PBS at a concentration of 4.8 mg/mL. Approximately 6,500 crystallization conditions were tested with the sitting-drop vapor diffusion method in 96-well plates. Irregularly shaped crystals with a longest dimension of ∼0.2 mm were obtained by mixing 150 nL of virus suspension with 50 nL of 0.8 M potassium dihydrogen phosphate, 0.8 M sodium dihydrogen phosphate, 0.1 M sodium Hepes, pH 7.5.

DWV Diffraction Data Collection, Structure Determination, and Refinement.

For data collection, DWV virion crystals were directly frozen in liquid nitrogen. The addition of a cryo-protectant was not necessary. Data were collected from a single crystal at 100 K at the Pilatus detector on beamline Proxima 1 at the Soleil synchrotron radiation source (Paris). An oscillation range of 0.1° was used during data collection. The crystal diffracted to a resolution of 3.8 Å. Data were processed and scaled using the package XDS (54).

The DWV virion crystals were of space group I23. Rotation function plots and packing considerations indicated that 1/12 of a virus particle (one pentamer of capsid protein protomers) occupied a crystallographic asymmetric unit. There are two possibilities for superimposing icosahedral 532 symmetry with the 23 symmetry of the crystal, which are perpendicular to each other. The orientation of the virion was determined from a plot of the fivefold rotation function, calculated with the program GLRF (55). Reflections between resolutions of 5.0 and 4.5 Å were used for the calculations. Because of the superposition of the icosahedral and crystallographic symmetry, the center of the particle had to be positioned at the intersection of the twofold and threefold symmetry axes of the crystal. The PDB structure of DWV, determined based on the cryo-EM reconstruction, was used for the molecular replacement. The model was placed into the above orientation and position in the unit cell and an initial low-resolution electron density map was calculated, suggesting that the P domain occupied a different position on the virus surface than in the cryo-EM reconstruction. Therefore, residues belonging to the P domain were removed from the model, and the position of the P domain was determined using a molecular replacement search as implemented in the program Phaser (56). The positions of the subunits and of the P domain were subjected to rigid-body refinement using the program suite Phenix (53). The new model was used to calculate phases to a resolution of 10 Å in the program CNS. The phases were refined by 25 cycles of averaging with the program AVE (57), using fivefold noncrystallographic symmetry. Phase extension was applied to obtain phases for higher resolution reflections. The addition of a small fraction of higher resolution data (one index at a time) was followed by five cycles of averaging. This procedure was repeated until phases were obtained for all of the reflections to a resolution of 3.8 Å.

The model was refined by manual rebuilding using the program Coot (52), alternating with coordinate refinement in the program Phenix (53). Other calculations were done using CCP4 (58). No water molecules were added to the model.

Cloning, Expression, and Crystallization of the DWV P Domain.

The nucleotide sequence of the DWV P domain (residues 263 to 419 of VP3) was cloned into the expression plasmid pET22T under the control of the T7 promoter. The vector provided an N-terminal His₆-Smt3 fusion tag. The target sequence in the plasmid was verified by sequencing. Autoinduction medium ZYP-5052 (59) was inoculated with Escherichia coli BL21(DE3) containing the above-mentioned plasmid. The culture was cultivated for 26 h at 37 °C with 250 RPM orbital shaking until the stationary phase of growth was reached. Cells from 1 L of media were collected by centrifugation and resuspended in 40 mL of lysis buffer (50 mM Tris, 100 mM NaCl, 1 mg/mL lysozyme, and 1% wt/vol octylthioglucoside, pH8). The cells were lysed at room temperature for 45 min, and the lysate was clarified by centrifugation at 20,000 × g at 4 °C for 10 min. The resulting supernatant was applied to a HisTrap HP column (GE Healthcare), and the protein of interest was eluted with 50 mM Tris and 100 mM sodium chloride buffer containing 350 mM imidazole. After overnight cleavage of the His₆-Smt3 tag using Ulp1ac protease and the removal of imidazole, the solution was loaded onto a HisTrap HP column. The protein of interest, recovered from flowthrough fractions, was loaded into a Superdex 26/60 75 prep grade (pg) column (GE Healthcare) for final purification and eluted with a buffer containing 50 mM Tris, 100 mM sodium chloride, pH8. The eluted protein was concentrated using centrifugal concentrators. The purity of the protein was verified on an SDS polyacrylamide gel, and the final concentration was 21 mg/mL (determined by A₂₈₀).

Crystals of the DWV P domain were obtained at 4 °C using the sitting-drop vapor diffusion technique, with a bottom solution containing 4.3 M sodium chloride and 0.1 M Hepes, pH 7.5. The drops were prepared by mixing 50 nL of bottom solution with 150 nL of DWV P domain suspension (21 mg/mL). Cuboid-shaped crystals formed within 2 weeks.

DWV P Domain Data Collection, Structure Determination, and Refinement.

For data collection, DWV P domain crystals of about 0.25 mm were fished out and immediately frozen in liquid nitrogen. Cryo-protectant was not necessary. Data were collected from a single crystal at 100 K at the Pilatus detector on beamline Proxima 1 at the Soleil synchrotron radiation source (Paris). An oscillation range of 0.1° was used during data collection. The crystal diffracted to a resolution of 1.5 Å. Data were processed and scaled using the package XDS (54).

The crystals were of space group C222. The crystallographic asymmetric unit contained a single P domain. The model of the P domain comprising residues 263 to 419, built based on the cryo-EM reconstruction map, was used as a search model for molecular replacement using the program Phaser (56). The model was subjected to automated rebuilding and refinement using the programs Arp/wArp and Refmac (60, 61). The automated model building resulted in a 90% complete model with the assigned sequence. The model was refined by manual rebuilding using the program Coot, alternating with coordinate refinement using the program Phenix (52, 56).

Structure and Sequence Analysis.

Multiple sequence alignments were carried out using the ClustalW server (www.ebi.ac.uk/Tools/msa/clustalw2/) (62). Figures were generated using the programs UCSF Chimera (63), and Pymol (The PyMOL Molecular Graphics System, Version 1.7.4; Schrödinger, LLC.). Structure-based pairwise alignments of biological protomers of various picornaviruses were prepared using the program VMD (64). The similarity score provided by VMD was used as an evolutionary distance to construct a nexus-format matrix file, which was converted into the phylogenetic tree and visualized with the program SplitsTree (65).

Data Deposition.

Cryo-EM maps of the DWV virions from different conditions were deposited into the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-4014, EMD-3574, EMD-3570, and EMD-3575, and the corresponding coordinates were deposited into the protein data bank (PDB) under PDB ID codes 5L8Q, 5MV5, 5MUP, and 5MV6. The crystal structures of the DWV virion and P domain were deposited under PDB ID codes 5G52 and 5G51.

Supplementary Material

Supplementary File

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Supplementary File

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Acknowledgments

We thank synchrotron “Soleil” Proxima-1 and beamline scientist Dr. Pierre Legrand for help with X-ray data collection and processing. This research was carried out under the project CEITEC 2020 (LQ1601), with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under National Sustainability Program II. Access to computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum, provided under the program “Projects of Large Infrastructure for Research, Development, and Innovations” Grant (LM2010005), is greatly appreciated. This work was supported by the IT4Innovations Centre of Excellence project Grant (CZ.1.05/1.1.00/02.0070), funded by the European Regional Development Fund and the national budget of the Czech Republic via the Research and Development for Innovations Operational Program, as well as the Czech Ministry of Education, Youth and Sports via the project Large Research, Development and Innovations Infrastructures Grant (LM2011033). The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Program Grant (FP/2007-2013)/ERC Grant Agreement 355855 and from EMBO Grant Agreement European Molecular Biology Organization (EMBO)-IG 3041 (to P.P.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Cryo-EM maps of the DWV virions from different conditions have been deposited in the Electron Microscopy Data Bank (EMDB) (accession nos. EMD-4014, EMD-3574, EMD-3570, and EMD-3575); the corresponding coordinates and structure factors have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID codes 5L8Q, 5MV5, 5MUP, and 5MV6). The crystal structures of the DWV virion and P domain have been deposited under PDB ID codes 5G52 and 5G51.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1615695114/-/DCSupplemental.

References

1.Smith KM, et al. Pathogens, pests, and economics: Drivers of honey bee colony declines and losses. EcoHealth. 2013;10(4):434–445. doi: 10.1007/s10393-013-0870-2. [DOI] [PubMed] [Google Scholar]
2.Allsopp MH, de Lange WJ, Veldtman R. Valuing insect pollination services with cost of replacement. PLoS One. 2008;3(9):e3128. doi: 10.1371/journal.pone.0003128. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Biesmeijer JC, et al. Parallel declines in pollinators and insect-pollinated plants in Britain and the Netherlands. Science. 2006;313(5785):351–354. doi: 10.1126/science.1127863. [DOI] [PubMed] [Google Scholar]
4.Dainat B, Vanengelsdorp D, Neumann P. Colony collapse disorder in Europe. Environ Microbiol Rep. 2012;4(1):123–125. doi: 10.1111/j.1758-2229.2011.00312.x. [DOI] [PubMed] [Google Scholar]
5.van Engelsdorp D, Hayes J, Jr, Underwood RM, Pettis J. A survey of honey bee colony losses in the U.S., fall 2007 to spring 2008. PLoS One. 2008;3(12):e4071. doi: 10.1371/journal.pone.0004071. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Bowen-Walker PL, Martin SJ, Gunn A. The transmission of deformed wing virus between honeybees (Apis mellifera L.) by the ectoparasitic mite varroa jacobsoni Oud. J Invertebr Pathol. 1999;73(1):101–106. doi: 10.1006/jipa.1998.4807. [DOI] [PubMed] [Google Scholar]
7.Shen M, Yang X, Cox-Foster D, Cui L. The role of varroa mites in infections of Kashmir bee virus (KBV) and deformed wing virus (DWV) in honey bees. Virology. 2005;342(1):141–149. doi: 10.1016/j.virol.2005.07.012. [DOI] [PubMed] [Google Scholar]
8.Highfield AC, et al. Deformed wing virus implicated in overwintering honeybee colony losses. Appl Environ Microbiol. 2009;75(22):7212–7220. doi: 10.1128/AEM.02227-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.de Miranda JR, Gauthier L, Ribiere M, Chen YP. Honey bee viruses and their effect on bee and colony health. In: Sammataro DYJ, editor. Honey Bee Colony Health: Challenges and Sustainable Solutions. CRC Press; Boca Raton, FL: 2012. pp. 71–102. [Google Scholar]
10.Le Gall O, et al. Picornavirales, a proposed order of positive-sense single-stranded RNA viruses with a pseudo-T = 3 virion architecture. Arch Virol. 2008;153(4):715–727. doi: 10.1007/s00705-008-0041-x. [DOI] [PubMed] [Google Scholar]
11.Lanzi G, et al. Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.) J Virol. 2006;80(10):4998–5009. doi: 10.1128/JVI.80.10.4998-5009.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Zhang J, et al. Three-dimensional structure of the Chinese Sacbrood bee virus. Sci China C Life Sci. 2001;44(4):443–448. doi: 10.1007/BF02879612. [DOI] [PubMed] [Google Scholar]
13.Kalynych S, et al. Virion structure of iflavirus slow bee paralysis virus at 2.6-angstrom resolution. J Virol. 2016;90(16):7444–7455. doi: 10.1128/JVI.00680-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.de Miranda JR, et al. Genetic characterization of slow bee paralysis virus of the honeybee (Apis mellifera L.) J Gen Virol. 2010;91(Pt 10):2524–2530. doi: 10.1099/vir.0.022434-0. [DOI] [PubMed] [Google Scholar]
15.Rossmann MG, et al. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature. 1985;317(6033):145–153. doi: 10.1038/317145a0. [DOI] [PubMed] [Google Scholar]
16.Harrison SC, Olson AJ, Schutt CE, Winkler FK, Bricogne G. Tomato bushy stunt virus at 2.9 A resolution. Nature. 1978;276(5686):368–373. doi: 10.1038/276368a0. [DOI] [PubMed] [Google Scholar]
17.Bergelson JM, Coyne CB. Picornavirus entry. Adv Exp Med Biol. 2013;790:24–41. doi: 10.1007/978-1-4614-7651-1_2. [DOI] [PubMed] [Google Scholar]
18.Fuchs R, Blaas D. Productive entry pathways of human rhinoviruses. Adv Virol. 2012;2012:826301. doi: 10.1155/2012/826301. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Jackson T, Mould AP, Sheppard D, King AM. Integrin alphavbeta1 is a receptor for foot-and-mouth disease virus. J Virol. 2002;76(3):935–941. doi: 10.1128/JVI.76.3.935-941.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Boonyakiat Y, Hughes PJ, Ghazi F, Stanway G. Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry. J Virol. 2001;75(20):10000–10004. doi: 10.1128/JVI.75.20.10000-10004.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Kalynych S, Pálková L, Plevka P. The structure of human parechovirus 1 reveals an association of the RNA genome with the capsid. J Virol. 2015;90(3):1377–1386. doi: 10.1128/JVI.02346-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Holm L, Rosenstrom P. Dali server: Conservation mapping in 3D. Nucleic Acids Res. 2010;38(Web Server issue):W545–W549. doi: 10.1093/nar/gkq366. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.York RL, et al. Structural, mechanistic, and antigenic characterization of the human astrovirus capsid. J Virol. 2015;90(5):2254–2263. doi: 10.1128/JVI.02666-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Dodson G, Wlodawer A. Catalytic triads and their relatives. Trends Biochem Sci. 1998;23(9):347–352. doi: 10.1016/s0968-0004(98)01254-7. [DOI] [PubMed] [Google Scholar]
25.Canaan S, Roussel A, Verger R, Cambillau C. Gastric lipase: Crystal structure and activity. Biochim Biophys Acta. 1999;1441(2-3):197–204. doi: 10.1016/s1388-1981(99)00160-2. [DOI] [PubMed] [Google Scholar]
26.Vaquero ME, Barriuso J, Martínez MJ, Prieto A. Properties, structure, and applications of microbial sterol esterases. Appl Microbiol Biotechnol. 2016;100(5):2047–2061. doi: 10.1007/s00253-015-7258-x. [DOI] [PubMed] [Google Scholar]
27.Ongus JR, et al. Complete sequence of a picorna-like virus of the genus Iflavirus replicating in the mite Varroa destructor. J Gen Virol. 2004;85(Pt 12):3747–3755. doi: 10.1099/vir.0.80470-0. [DOI] [PubMed] [Google Scholar]
28.Fujiyuki T, et al. Novel insect picorna-like virus identified in the brains of aggressive worker honeybees. J Virol. 2004;78(3):1093–1100. doi: 10.1128/JVI.78.3.1093-1100.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Bailey L, Woods RD. Three previously undescribed viruses from the honey bee. J Gen Virol. 1974;25(2):175–186. doi: 10.1099/0022-1317-25-2-175. [DOI] [PubMed] [Google Scholar]
30.Cao S, et al. Structural basis for the recognition of blood group trisaccharides by norovirus. J Virol. 2007;81(11):5949–5957. doi: 10.1128/JVI.00219-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Hansman GS, et al. Structural basis for norovirus inhibition and fucose mimicry by citrate. J Virol. 2012;86(1):284–292. doi: 10.1128/JVI.05909-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Farr GA, Zhang LG, Tattersall P. Parvoviral virions deploy a capsid-tethered lipolytic enzyme to breach the endosomal membrane during cell entry. Proc Natl Acad Sci USA. 2005;102(47):17148–17153. doi: 10.1073/pnas.0508477102. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Mullapudi E, Přidal A, Pálková L, de Miranda JR, Plevka P. Virion structure of Israeli acute bee paralysis virus. J Virol. 2016;90(18):8150–8159. doi: 10.1128/JVI.00854-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Tate J, et al. The crystal structure of cricket paralysis virus: The first view of a new virus family. Nat Struct Biol. 1999;6(8):765–774. doi: 10.1038/11543. [DOI] [PubMed] [Google Scholar]
35.Squires G, et al. Structure of the triatoma virus capsid. Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 6):1026–1037. doi: 10.1107/S0907444913004617. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Wang X, et al. Hepatitis A virus and the origins of picornaviruses. Nature. 2015;517(7532):85–88. doi: 10.1038/nature13806. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Smith TJ, et al. The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating. Science. 1986;233(4770):1286–1293. doi: 10.1126/science.3018924. [DOI] [PubMed] [Google Scholar]
38.Hadfield AT, Diana GD, Rossmann MG. Analysis of three structurally related antiviral compounds in complex with human rhinovirus 16. Proc Natl Acad Sci USA. 1999;96(26):14730–14735. doi: 10.1073/pnas.96.26.14730. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Grant RA, et al. Structures of poliovirus complexes with anti-viral drugs: Implications for viral stability and drug design. Curr Biol. 1994;4(9):784–797. doi: 10.1016/s0960-9822(00)00176-7. [DOI] [PubMed] [Google Scholar]
40.Morgunova EYu, et al. The atomic structure of carnation mottle virus capsid protein. FEBS Lett. 1994;338(3):267–271. doi: 10.1016/0014-5793(94)80281-5. [DOI] [PubMed] [Google Scholar]
41.Zhu L, et al. Structure of Ljungan virus provides insight into genome packaging of this picornavirus. Nat Commun. 2015;6:8316. doi: 10.1038/ncomms9316. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Shakeel S, et al. Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis. Nat Commun. 2016;7:11387. doi: 10.1038/ncomms11387. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Nugent CI, Kirkegaard K. RNA binding properties of poliovirus subviral particles. J Virol. 1995;69(1):13–22. doi: 10.1128/jvi.69.1.13-22.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.de Miranda JR, et al. Standard methods for Apis mellifera pest and pathogen research. In: Dietemann V, Ellis JD, Neumann P, editors. The COLOSS BEEBOOK. Vol II IBRA; Treforest, UK: 2013. [Google Scholar]
45.Scheres SH. RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol. 2012;180(3):519–530. doi: 10.1016/j.jsb.2012.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Davis IW, et al. MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 2007;35(Web Server issue):W375–W383. doi: 10.1093/nar/gkm216. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Shaikh TR, et al. SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat Protoc. 2008;3(12):1941–1974. doi: 10.1038/nprot.2008.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Tang G, et al. EMAN2: An extensible image processing suite for electron microscopy. J Struct Biol. 2007;157(1):38–46. doi: 10.1016/j.jsb.2006.05.009. [DOI] [PubMed] [Google Scholar]
49.Mindell JA, Grigorieff N. Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol. 2003;142(3):334–347. doi: 10.1016/s1047-8477(03)00069-8. [DOI] [PubMed] [Google Scholar]
50.Scheres SH, Chen S. Prevention of overfitting in cryo-EM structure determination. Nat Methods. 2012;9(9):853–854. doi: 10.1038/nmeth.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Chen S, et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy. 2013;135:24–35. doi: 10.1016/j.ultramic.2013.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Emsley P, Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 12 Pt 1):2126–2132. doi: 10.1107/S0907444904019158. [DOI] [PubMed] [Google Scholar]
53.Afonine PV, et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr. 2012;68(Pt 4):352–367. doi: 10.1107/S0907444912001308. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Kabsch W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 2):133–144. doi: 10.1107/S0907444909047374. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Tong L, Rossmann MG. Rotation function calculations with GLRF program. Methods Enzymol. 1997;276:594–611. [PubMed] [Google Scholar]
56.McCoy AJ, et al. Phaser crystallographic software. J Appl Cryst. 2007;40(Pt 4):658–674. doi: 10.1107/S0021889807021206. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Kleywegt GJ, Read RJ. Not your average density. Structure. 1997;5(12):1557–1569. doi: 10.1016/s0969-2126(97)00305-5. [DOI] [PubMed] [Google Scholar]
58.Winn MD, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 4):235–242. doi: 10.1107/S0907444910045749. [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005;41(1):207–234. doi: 10.1016/j.pep.2005.01.016. [DOI] [PubMed] [Google Scholar]
60.Vagin AA, et al. REFMAC5 dictionary: Organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 12 Pt 1):2184–2195. doi: 10.1107/S0907444904023510. [DOI] [PubMed] [Google Scholar]
61.Langer G, Cohen SX, Lamzin VS, Perrakis A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat Protoc. 2008;3(7):1171–1179. doi: 10.1038/nprot.2008.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Chenna R, et al. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 2003;31(13):3497–3500. doi: 10.1093/nar/gkg500. [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Pettersen EF, et al. UCSF Chimera: A visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–1612. doi: 10.1002/jcc.20084. [DOI] [PubMed] [Google Scholar]
64.Humphrey W, Dalke A, Schulten K. VMD: Visual molecular dynamics. J Mol Graph. 1996;14(1):33–38. doi: 10.1016/0263-7855(96)00018-5. [DOI] [PubMed] [Google Scholar]
65.Huson DH. SplitsTree: Analyzing and visualizing evolutionary data. Bioinformatics. 1998;14(1):68–73. doi: 10.1093/bioinformatics/14.1.68. [DOI] [PubMed] [Google Scholar]

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[r1] 1.Smith KM, et al. Pathogens, pests, and economics: Drivers of honey bee colony declines and losses. EcoHealth. 2013;10(4):434–445. doi: 10.1007/s10393-013-0870-2. [DOI] [PubMed] [Google Scholar]

[r2] 2.Allsopp MH, de Lange WJ, Veldtman R. Valuing insect pollination services with cost of replacement. PLoS One. 2008;3(9):e3128. doi: 10.1371/journal.pone.0003128. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r3] 3.Biesmeijer JC, et al. Parallel declines in pollinators and insect-pollinated plants in Britain and the Netherlands. Science. 2006;313(5785):351–354. doi: 10.1126/science.1127863. [DOI] [PubMed] [Google Scholar]

[r4] 4.Dainat B, Vanengelsdorp D, Neumann P. Colony collapse disorder in Europe. Environ Microbiol Rep. 2012;4(1):123–125. doi: 10.1111/j.1758-2229.2011.00312.x. [DOI] [PubMed] [Google Scholar]

[r5] 5.van Engelsdorp D, Hayes J, Jr, Underwood RM, Pettis J. A survey of honey bee colony losses in the U.S., fall 2007 to spring 2008. PLoS One. 2008;3(12):e4071. doi: 10.1371/journal.pone.0004071. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r6] 6.Bowen-Walker PL, Martin SJ, Gunn A. The transmission of deformed wing virus between honeybees (Apis mellifera L.) by the ectoparasitic mite varroa jacobsoni Oud. J Invertebr Pathol. 1999;73(1):101–106. doi: 10.1006/jipa.1998.4807. [DOI] [PubMed] [Google Scholar]

[r7] 7.Shen M, Yang X, Cox-Foster D, Cui L. The role of varroa mites in infections of Kashmir bee virus (KBV) and deformed wing virus (DWV) in honey bees. Virology. 2005;342(1):141–149. doi: 10.1016/j.virol.2005.07.012. [DOI] [PubMed] [Google Scholar]

[r8] 8.Highfield AC, et al. Deformed wing virus implicated in overwintering honeybee colony losses. Appl Environ Microbiol. 2009;75(22):7212–7220. doi: 10.1128/AEM.02227-09. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r9] 9.de Miranda JR, Gauthier L, Ribiere M, Chen YP. Honey bee viruses and their effect on bee and colony health. In: Sammataro DYJ, editor. Honey Bee Colony Health: Challenges and Sustainable Solutions. CRC Press; Boca Raton, FL: 2012. pp. 71–102. [Google Scholar]

[r10] 10.Le Gall O, et al. Picornavirales, a proposed order of positive-sense single-stranded RNA viruses with a pseudo-T = 3 virion architecture. Arch Virol. 2008;153(4):715–727. doi: 10.1007/s00705-008-0041-x. [DOI] [PubMed] [Google Scholar]

[r11] 11.Lanzi G, et al. Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.) J Virol. 2006;80(10):4998–5009. doi: 10.1128/JVI.80.10.4998-5009.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r12] 12.Zhang J, et al. Three-dimensional structure of the Chinese Sacbrood bee virus. Sci China C Life Sci. 2001;44(4):443–448. doi: 10.1007/BF02879612. [DOI] [PubMed] [Google Scholar]

[r13] 13.Kalynych S, et al. Virion structure of iflavirus slow bee paralysis virus at 2.6-angstrom resolution. J Virol. 2016;90(16):7444–7455. doi: 10.1128/JVI.00680-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r14] 14.de Miranda JR, et al. Genetic characterization of slow bee paralysis virus of the honeybee (Apis mellifera L.) J Gen Virol. 2010;91(Pt 10):2524–2530. doi: 10.1099/vir.0.022434-0. [DOI] [PubMed] [Google Scholar]

[r15] 15.Rossmann MG, et al. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature. 1985;317(6033):145–153. doi: 10.1038/317145a0. [DOI] [PubMed] [Google Scholar]

[r16] 16.Harrison SC, Olson AJ, Schutt CE, Winkler FK, Bricogne G. Tomato bushy stunt virus at 2.9 A resolution. Nature. 1978;276(5686):368–373. doi: 10.1038/276368a0. [DOI] [PubMed] [Google Scholar]

[r17] 17.Bergelson JM, Coyne CB. Picornavirus entry. Adv Exp Med Biol. 2013;790:24–41. doi: 10.1007/978-1-4614-7651-1_2. [DOI] [PubMed] [Google Scholar]

[r18] 18.Fuchs R, Blaas D. Productive entry pathways of human rhinoviruses. Adv Virol. 2012;2012:826301. doi: 10.1155/2012/826301. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r19] 19.Jackson T, Mould AP, Sheppard D, King AM. Integrin alphavbeta1 is a receptor for foot-and-mouth disease virus. J Virol. 2002;76(3):935–941. doi: 10.1128/JVI.76.3.935-941.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r20] 20.Boonyakiat Y, Hughes PJ, Ghazi F, Stanway G. Arginine-glycine-aspartic acid motif is critical for human parechovirus 1 entry. J Virol. 2001;75(20):10000–10004. doi: 10.1128/JVI.75.20.10000-10004.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r21] 21.Kalynych S, Pálková L, Plevka P. The structure of human parechovirus 1 reveals an association of the RNA genome with the capsid. J Virol. 2015;90(3):1377–1386. doi: 10.1128/JVI.02346-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r22] 22.Holm L, Rosenstrom P. Dali server: Conservation mapping in 3D. Nucleic Acids Res. 2010;38(Web Server issue):W545–W549. doi: 10.1093/nar/gkq366. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r23] 23.York RL, et al. Structural, mechanistic, and antigenic characterization of the human astrovirus capsid. J Virol. 2015;90(5):2254–2263. doi: 10.1128/JVI.02666-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r24] 24.Dodson G, Wlodawer A. Catalytic triads and their relatives. Trends Biochem Sci. 1998;23(9):347–352. doi: 10.1016/s0968-0004(98)01254-7. [DOI] [PubMed] [Google Scholar]

[r25] 25.Canaan S, Roussel A, Verger R, Cambillau C. Gastric lipase: Crystal structure and activity. Biochim Biophys Acta. 1999;1441(2-3):197–204. doi: 10.1016/s1388-1981(99)00160-2. [DOI] [PubMed] [Google Scholar]

[r26] 26.Vaquero ME, Barriuso J, Martínez MJ, Prieto A. Properties, structure, and applications of microbial sterol esterases. Appl Microbiol Biotechnol. 2016;100(5):2047–2061. doi: 10.1007/s00253-015-7258-x. [DOI] [PubMed] [Google Scholar]

[r27] 27.Ongus JR, et al. Complete sequence of a picorna-like virus of the genus Iflavirus replicating in the mite Varroa destructor. J Gen Virol. 2004;85(Pt 12):3747–3755. doi: 10.1099/vir.0.80470-0. [DOI] [PubMed] [Google Scholar]

[r28] 28.Fujiyuki T, et al. Novel insect picorna-like virus identified in the brains of aggressive worker honeybees. J Virol. 2004;78(3):1093–1100. doi: 10.1128/JVI.78.3.1093-1100.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r29] 29.Bailey L, Woods RD. Three previously undescribed viruses from the honey bee. J Gen Virol. 1974;25(2):175–186. doi: 10.1099/0022-1317-25-2-175. [DOI] [PubMed] [Google Scholar]

[r30] 30.Cao S, et al. Structural basis for the recognition of blood group trisaccharides by norovirus. J Virol. 2007;81(11):5949–5957. doi: 10.1128/JVI.00219-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r31] 31.Hansman GS, et al. Structural basis for norovirus inhibition and fucose mimicry by citrate. J Virol. 2012;86(1):284–292. doi: 10.1128/JVI.05909-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r32] 32.Farr GA, Zhang LG, Tattersall P. Parvoviral virions deploy a capsid-tethered lipolytic enzyme to breach the endosomal membrane during cell entry. Proc Natl Acad Sci USA. 2005;102(47):17148–17153. doi: 10.1073/pnas.0508477102. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r33] 33.Mullapudi E, Přidal A, Pálková L, de Miranda JR, Plevka P. Virion structure of Israeli acute bee paralysis virus. J Virol. 2016;90(18):8150–8159. doi: 10.1128/JVI.00854-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r34] 34.Tate J, et al. The crystal structure of cricket paralysis virus: The first view of a new virus family. Nat Struct Biol. 1999;6(8):765–774. doi: 10.1038/11543. [DOI] [PubMed] [Google Scholar]

[r35] 35.Squires G, et al. Structure of the triatoma virus capsid. Acta Crystallogr D Biol Crystallogr. 2013;69(Pt 6):1026–1037. doi: 10.1107/S0907444913004617. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r36] 36.Wang X, et al. Hepatitis A virus and the origins of picornaviruses. Nature. 2015;517(7532):85–88. doi: 10.1038/nature13806. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r37] 37.Smith TJ, et al. The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating. Science. 1986;233(4770):1286–1293. doi: 10.1126/science.3018924. [DOI] [PubMed] [Google Scholar]

[r38] 38.Hadfield AT, Diana GD, Rossmann MG. Analysis of three structurally related antiviral compounds in complex with human rhinovirus 16. Proc Natl Acad Sci USA. 1999;96(26):14730–14735. doi: 10.1073/pnas.96.26.14730. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r39] 39.Grant RA, et al. Structures of poliovirus complexes with anti-viral drugs: Implications for viral stability and drug design. Curr Biol. 1994;4(9):784–797. doi: 10.1016/s0960-9822(00)00176-7. [DOI] [PubMed] [Google Scholar]

[r40] 40.Morgunova EYu, et al. The atomic structure of carnation mottle virus capsid protein. FEBS Lett. 1994;338(3):267–271. doi: 10.1016/0014-5793(94)80281-5. [DOI] [PubMed] [Google Scholar]

[r41] 41.Zhu L, et al. Structure of Ljungan virus provides insight into genome packaging of this picornavirus. Nat Commun. 2015;6:8316. doi: 10.1038/ncomms9316. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r42] 42.Shakeel S, et al. Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis. Nat Commun. 2016;7:11387. doi: 10.1038/ncomms11387. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r43] 43.Nugent CI, Kirkegaard K. RNA binding properties of poliovirus subviral particles. J Virol. 1995;69(1):13–22. doi: 10.1128/jvi.69.1.13-22.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r44] 44.de Miranda JR, et al. Standard methods for Apis mellifera pest and pathogen research. In: Dietemann V, Ellis JD, Neumann P, editors. The COLOSS BEEBOOK. Vol II IBRA; Treforest, UK: 2013. [Google Scholar]

[r45] 45.Scheres SH. RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol. 2012;180(3):519–530. doi: 10.1016/j.jsb.2012.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r46] 46.Davis IW, et al. MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 2007;35(Web Server issue):W375–W383. doi: 10.1093/nar/gkm216. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r47] 47.Shaikh TR, et al. SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat Protoc. 2008;3(12):1941–1974. doi: 10.1038/nprot.2008.156. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r48] 48.Tang G, et al. EMAN2: An extensible image processing suite for electron microscopy. J Struct Biol. 2007;157(1):38–46. doi: 10.1016/j.jsb.2006.05.009. [DOI] [PubMed] [Google Scholar]

[r49] 49.Mindell JA, Grigorieff N. Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol. 2003;142(3):334–347. doi: 10.1016/s1047-8477(03)00069-8. [DOI] [PubMed] [Google Scholar]

[r50] 50.Scheres SH, Chen S. Prevention of overfitting in cryo-EM structure determination. Nat Methods. 2012;9(9):853–854. doi: 10.1038/nmeth.2115. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r51] 51.Chen S, et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy. 2013;135:24–35. doi: 10.1016/j.ultramic.2013.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r52] 52.Emsley P, Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 12 Pt 1):2126–2132. doi: 10.1107/S0907444904019158. [DOI] [PubMed] [Google Scholar]

[r53] 53.Afonine PV, et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr. 2012;68(Pt 4):352–367. doi: 10.1107/S0907444912001308. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r54] 54.Kabsch W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 2):133–144. doi: 10.1107/S0907444909047374. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r55] 55.Tong L, Rossmann MG. Rotation function calculations with GLRF program. Methods Enzymol. 1997;276:594–611. [PubMed] [Google Scholar]

[r56] 56.McCoy AJ, et al. Phaser crystallographic software. J Appl Cryst. 2007;40(Pt 4):658–674. doi: 10.1107/S0021889807021206. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r57] 57.Kleywegt GJ, Read RJ. Not your average density. Structure. 1997;5(12):1557–1569. doi: 10.1016/s0969-2126(97)00305-5. [DOI] [PubMed] [Google Scholar]

[r58] 58.Winn MD, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 4):235–242. doi: 10.1107/S0907444910045749. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r59] 59.Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005;41(1):207–234. doi: 10.1016/j.pep.2005.01.016. [DOI] [PubMed] [Google Scholar]

[r60] 60.Vagin AA, et al. REFMAC5 dictionary: Organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 12 Pt 1):2184–2195. doi: 10.1107/S0907444904023510. [DOI] [PubMed] [Google Scholar]

[r61] 61.Langer G, Cohen SX, Lamzin VS, Perrakis A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat Protoc. 2008;3(7):1171–1179. doi: 10.1038/nprot.2008.91. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r62] 62.Chenna R, et al. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 2003;31(13):3497–3500. doi: 10.1093/nar/gkg500. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r63] 63.Pettersen EF, et al. UCSF Chimera: A visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–1612. doi: 10.1002/jcc.20084. [DOI] [PubMed] [Google Scholar]

[r64] 64.Humphrey W, Dalke A, Schulten K. VMD: Visual molecular dynamics. J Mol Graph. 1996;14(1):33–38. doi: 10.1016/0263-7855(96)00018-5. [DOI] [PubMed] [Google Scholar]

[r65] 65.Huson DH. SplitsTree: Analyzing and visualizing evolutionary data. Bioinformatics. 1998;14(1):68–73. doi: 10.1093/bioinformatics/14.1.68. [DOI] [PubMed] [Google Scholar]

PERMALINK

Structure of deformed wing virus, a major honey bee pathogen

Karel Škubník

Jiří Nováček

Tibor Füzik

Antonín Přidal

Robert J Paxton

Pavel Plevka

Significance

Abstract

Results and Discussion

Structure of DWV Virion and Capsid Proteins.

Table S1.

Fig. S1.

Fig. 1.

Structure of the P Domain.

Fig. 2.

Comparison of DWV and SBPV Virion Structures.

Fig. S2.

Comparison of the P Domain with Other Proteins.

Table S2.

Position of the P Domain at the Virion Surface.

Fig. S3.

Fig. S4.

Fig. S5.

Putative Role of the P Domain in Cell Entry.

Fig. 3.

Evolutionary Relationship to Other Viruses from the Order Picornavirales.

Fig. 4.

Capsid–RNA Interactions.

Fig. 5.

Materials and Methods

SI Materials and Methods

Virus Propagation in Honey Bee Pupae.

Virus Purification.

Single Particle Data Acquisition and Image Processing.

Icosahedral Reconstruction of the DWV Virion.

Cryo-EM Structure Determination and Refinement.

Crystallization of the DWV Virion.

DWV Diffraction Data Collection, Structure Determination, and Refinement.

Cloning, Expression, and Crystallization of the DWV P Domain.

DWV P Domain Data Collection, Structure Determination, and Refinement.

Structure and Sequence Analysis.

Data Deposition.

Supplementary Material

Acknowledgments

Footnotes

References

Associated Data

Supplementary Materials

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