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. 2017 Feb 28;91(6):e02100-16.
doi: 10.1128/JVI.02100-16. Print 2017 Mar 15.

Virion Structure of Black Queen Cell Virus, a Common Honeybee Pathogen

Radovan Spurny  1 , Antonín Přidal  2 , Lenka Pálková  1 , Hoa Khanh Tran Kiem  1 , Joachim R de Miranda  3 , Pavel Plevka  4
Affiliations

Virion Structure of Black Queen Cell Virus, a Common Honeybee Pathogen

Radovan Spurny et al. J Virol. .

Abstract

Viral diseases are a major threat to honeybee (Apis mellifera) populations worldwide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae, which is part of the order Picornavirales Here we present a crystal structure of BQCV determined to a resolution of 3.4 Å. The virion is formed by 60 copies of each of the major capsid proteins VP1, VP2, and VP3; however, there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae The C terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-Å-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses.IMPORTANCE The western honeybee is the most important pollinator of all, and it is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV, determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.

Keywords: Apis mellifera; Cripavirus; Dicistroviridae; Picornavirales; Triatovirus; X ray; X-ray crystallography; capsid; crystallography; honey bee; honeybee; insect disease; structure; virion; virus.

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Figures

FIG 1
FIG 1
Comparison of virion structures of BQCV, TrV, CrPV, and IAPV. Molecular surfaces of BQCV (A), TrV (B), CrPV (C), and IAPV (D) virions are rainbow-colored based on their distance from the virion center. Depressions are shown in blue and protrusions in red.
FIG 2
FIG 2
Comparison of structures of icosahedral asymmetric units of BQCV, TrV, CrPV, and IAPV. Shown are cartoon representations of the capsid protein protomers of BQCV (A), TrV (B), CrPV (C), and IAPV (D). VP1 subunits are shown in blue, VP2 in green, VP3 in red, and VP4 (if present) in yellow. Names of the β-strands of the capsid proteins are shown. The positions of the 5-fold, 3-fold, and 2-fold icosahedral symmetry axes are indicated with pentagons, triangles, and ovals, respectively.
FIG 3
FIG 3
Comparison of prominent virion surface features of BQCV, TrV, CrPV, and IAPV. (A) Cross section of capsids close to 5-fold icosahedral axes are shown in gray. Cartoon representations of capsid proteins from a selected icosahedral asymmetric unit are shown in blue for VP1, green for VP2, red for VP3, and yellow for VP4. Finger-like protrusions of BQCV formed by the C terminus of VP1 and CD loops of VP1 and VP3 are larger than those of TrV, CrPV, and IAPV. The positions of the 5-fold icosahedral symmetry axes are indicated with dashed lines. (B) Comparison of VP1 subunits is shown. The CD loops are highlighted in red, the EF loops in orange, and the C termini in green. Names of the secondary structure elements are indicated. (C) Comparison of VP3 subunits. The CD loops of VP3 are highlighted in cyan, the GH loops in green, and the EF loops in magenta.
FIG 4
FIG 4
Maps of electron densities of capsids of dicistroviruses close to icosahedral 5-fold axes. Electron densities attributed to putative ions are present on 5-fold axes of BQCV (A) and TrV (B). In contrast, the density is absent in CrPV (C) and IAPV (D). The density maps are shown as gray meshes contoured at 1.8 σ. VP1 subunits are shown in stick representation with carbon atoms in blue. The names of residues of BQCV and TrV closest to the putative ion densities are shown.
FIG 5
FIG 5
Negative-stain electron microscopy picture of BQCV after purification on CsCl gradient. See Materials and Methods for details on the purification procedure.
FIG 6
FIG 6
Putative proteolytic site in VP1 subunits of dicistroviruses. The residues Asp-Asp-Phe/Met of VP1 that were speculated to mediate the cleavage of VP0 into VP3 and VP4 are positioned close to the N terminus of VP3 and C terminus of VP4 from another protomer related by an icosahedral 5-fold axis of symmetry. The residues constituting the putative active site are shown in stick representation. VP1 subunits are shown in blue and VP3 in red.
FIG 7
FIG 7
BQCV crystals contain VP4 subunits and the crystallized virus is infectious. (A) Polyacrylamide gel electrophoresis of capsid proteins of BQCV. Lane 1, marker; lane 2, purified BQCV; lane 3, BQCV dissolved from crystals. Arrowhead and VP4 label indicate the position of capsid protein VP4 (8.1 kDa). Capsid proteins VP1, VP2, and VP3 of BQCV have molecular masses in the 25- to 35-kDa range. (B) Agarose gel electrophoresis of PCR fragments obtained from reverse-transcribed RNA isolated from pupae injected with native BQCV (lane 2), BQCV dissolved from crystals (lane 3), and mock-infected with PBS (lane 4). Please see Materials and Methods for details. Lane 1, DNA ladder. (C to H) Images of pupae injected with BQCV dissolved from crystals (C and D) or native virus (E and F) or mock infected with PBS (G and H). The pupae were imaged 1 day (C, E, and G) and 5 days (D, F, and H) after the injection. The pupae injected with virus (C to F) developed slower than the mock-injected pupae (G and H), as shown by the delay in color development of the eyes and the darkening of the body 5 days postinfection. Two pupae missing in the panels (C and D) were accidentally destroyed during imaging.
FIG 8
FIG 8
VP1 of BQCV does not contain a hydrophobic pocket. VP1 of BQCV (A) and human enterovirus 71 (EV71) (B) are shown in cartoon representations. The pocket factor of human enterovirus 71 is shown as a stick model in green. The volume of the pocket calculated with the program Caver is shown in panel B. In addition, the side chains of residues that interact with the pocket factor are shown as sticks. In BQCV, the core of the VP1 subunits is filled by side chains of residues forming the β-sheet BIDG and CHEF. The residues Asn71 and Tyr116 in BQCV obscure the volume that corresponds to the opening of the pocket at the capsid surface in EV71.
FIG 9
FIG 9
Evolutionary relationship among viruses from the Dicistroviridae, Picornaviridae, and Iflaviridae families based on structural alignment of capsid proteins. (A) Phylogenetic tree based on structural similarity of icosahedral asymmetric units of indicated viruses. (B) Evolutionary tree of dicistroviruses based on alignments of ORF2 sequences verifies division of dicistroviruses into genera Aparavirus, Cripavirus, and Triatovirus. For details on the construction of the diagram, please see Materials and Methods.
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