Structure of an enteric pathogen, bovine parvovirus

Abstract

Bovine parvovirus (BPV), the causative agent of respiratory and gastrointestinal disease in cows, is the type member of the Bocaparvovirus genus of the Parvoviridae family. Toward efforts to obtain a template for the development of vaccines and small-molecule inhibitors for this pathogen, the structure of the BPV capsid, assembled from the major capsid viral protein 2 (VP2), was determined using X-ray crystallography as well as cryo-electron microscopy and three-dimensional image reconstruction (cryo-reconstruction) to 3.2- and 8.8-Å resolutions, respectively. The VP2 region ordered in the crystal structure, from residues 39 to 536, conserves the parvoviral eight-stranded jellyroll motif and an αA helix. The BPV capsid displays common parvovirus features: a channel at and depressions surrounding the 5-fold axes and protrusions surrounding the 3-fold axes. However, rather than a depression centered at the 2-fold axes, a raised surface loop divides this feature in BPV. Additional observed density in the capsid interior in the cryo-reconstructed map, compared to the crystal structure, is interpreted as 10 additional N-terminal residues, residues 29 to 38, that radially extend the channel under the 5-fold axis, as observed for human bocavirus 1 (HBoV1). Surface loops of various lengths and conformations extend from the core jellyroll motif of VP2. These loops confer the unique surface topology of the BPV capsid, making it strikingly different from HBoV1 as well as the type members of other Parvovirinae genera for which structures have been determined. For the type members, regions structurally analogous to those decorating the BPV capsid surface serve as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity.

Importance: Bovine parvovirus (BPV), identified in the 1960s in diarrheic calves, is the type member of the Bocaparvovirus genus of the nonenveloped, single-stranded DNA (ssDNA) Parvoviridae family. The recent isolation of human bocaparvoviruses from children with severe respiratory and gastrointestinal infections has generated interest in understanding the life cycle and pathogenesis of these emerging viruses. We have determined the high-resolution structure of the BPV capsid assembled from its predominant capsid protein VP2, known to be involved in a myriad of functions during host cell entry, pathogenesis, and antigenicity for other members of the Parvovirinae. Our results show the conservation of the core secondary structural elements and the location of the N-terminal residues for the known bocaparvovirus capsid structures. However, surface loops with high variability in sequence and conformation give BPV a unique capsid surface topology. Similar analogous regions in other Parvovirinae type members are important as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity.

FIG 1

Purification, crystallization, and freezing of BPV VLPs. (A) Micrograph of negatively stained BPV VLPs imaged at a magnification of ×97,000. (B) Flat crystals of BPV (indicated by white arrows) obtained using a solution containing 10 mM Tris (pH 7.5), 4% PEG 8000, 8 mM MgCl₂, and 25 mM LiSO₄. (C) Cryomicrograph of unstained BPV VLPs.

FIG 2

Crystal structure of BPV. (A and B) Sections of the 2F_o-F_c electron density map, contoured at a threshold of 1.5 σ (gray mesh), highlighting the unbiased BPV structure at the residue (A) and polypeptide chain (B) levels. The stick and cartoon representations show the superposed coordinates of BPV (magenta) and B19 (orange). (C) Cartoon representation of the BPV VP2 crystal structure (magenta) with the following secondary structure elements highlighted: β-strands (red), loops (magenta), and helices (cyan). The conserved core consisting of the β-strands (βA-BIDG-CHEF) and α-helix (αA) is labeled accordingly. The approximate positions of the icosahedral 2-, 3-, and 5-fold symmetry axes are indicated as a filled oval, triangle, and pentagon, respectively. The N and C termini of the polypeptide chain are also labeled. (D) Radially depth-cued surface representation of the BPV capsid shown approximately along the 2-fold axis of symmetry. The approximate positions of the 2-, 3-, and 5-fold axes as well as the 2/5-fold wall are indicated. A color key corresponding to the depth cue radii (in Å) is shown. Panels A to C were generated with PyMOL, and panel D was generated with UCSF-Chimera (39, 40).

FIG 3

Structural alignment of BPV and HBoV1. Secondary structure elements, β-strands and α-helices, are indicated with arrows (blue) and cylinders (red), respectively. The conserved core elements (βA-BIDG-CHEF and αA) are labeled. Insertions, deletions, or VRs (as defined in Materials and Methods) are shown in red in the HBoV1 sequence and labeled. The first N-terminal residues modeled into the BPV reconstructed and crystal structures are indicated with black arrows. Symbols below the alignment indicate standard ClustalW2 nomenclature, where * indicates identity, : indicates high-level conservation, and · indicates low-level conservation.

FIG 4

Cryo-EM structure of BPV. (A) Surface representation of the cryo-reconstructed density map, viewed along the 2-fold axis, radially depth cued as per the color key (in units of Å). An equilateral triangle depicting the viral asymmetric unit, with the 3-fold axes at the base vertices separated by the 2-fold axis and the 5-fold axis at its vertex (labeled 3f, 2f, and 5f, respectively), is shown. (B) Zoom-in view of the conserved α-helix (αA) from 2-fold-related monomers showing the fit into the reconstructed density (gray mesh). (C) Cross-sectional view of the 60-mer of the BPV crystal structure docked into the cryo-reconstructed density map (shown as gray mesh and contoured at 1 σ). The dashed lines indicate the icosahedral symmetry axes. (D) Side view of a cross section of the map and fitted crystal structure (shown in rainbow colors), highlighting the extended 5-fold channel. The first N-terminal residues observed in the crystal structure and in the pseudoatomic BPV model built into the cryo-reconstructed map are indicated. Approximate dimensions for the width and height of the channel are also shown. This figure was generated using UCSF-Chimera (39).

FIG 5

Comparison of Bocaparvovirus cryo-EM structures. (A and B) Surface (A) and cross-sectional (B) views of BPV and previously reported HBoV1 (17) radially depth cued by the color key (in units of Å). (C) Superposition of the C_α of the crystal structure of BPV (magenta) and the revised pseudoatomic model of HBoV1 (red), depicted as smooth loops. The approximate positions of the icosahedral 2-, 3-, and 5-fold symmetry axes are indicated as a filled oval, triangle, and pentagon, respectively. The following VRs are labeled on the VP2 monomer (left) and colored on the surface of the BPV capsid (right) (in gray) in different colors: VR-II (blue), VR-III (yellow), VR-IV (red), VR-VIII (green), VR-VIIIB (wheat), and VR-IX (chocolate). Panels A and B were generated using UCSF-Chimera, and panel C was generated with PyMOL (39, 40).

FIG 6

Superposition of Parvovirinae VP2 structures and identification of VRs. (A) Cartoon representation of superposed atomic coordinates of B19 (orange), AAV2 (royal blue), and MVMp (green) as well as pseudoatomic coordinates of AMDV (cyan) with the BPV crystal structure (magenta). The positions of the icosahedral 2-fold, 3-fold, and 5-fold axes of symmetry are labeled as described in the legend of Fig. 2C. (B) Cartoon diagram (gray) of BPV with the following VRs: VR-I (purple), VR-II (blue), VR-IIA (forest green), VR-III (yellow), VR-IV (red), VR-V (gray), VR-VI (pink), VR-VII (cyan), VR-VIII (green), VR-VIIIA (orange), VR-VIIIB (wheat), and VR-IX (chocolate). (C) Surface representation of BPV capsid (gray) with VRs colored as described above for panel B. This figure was generated with PyMOL (40).

FIG 7

Comparison of Parvovirinae capsids. Shown are radially depth-cued surface representations (in units of Å) of the parvovirus capsids viewed along the icosahedral 2-fold axis of symmetry. These images were generated using the atomic coordinates of BPV, AAV2, MVMp, and B19 or pseudoatomic coordinates built into the cryo-reconstructed density of AMDV. This figure was generated with UCSF-Chimera (39).