Structural Characterization of Canine Minute Virus, Rat and Porcine Bocavirus

Abstract

Bocaparvovirus is an expansive genus of the Parvovirinae, with a wide range of vertebrate hosts. This study investigates Canine minute virus (CnMV), Rat bocavirus (RBoV), and Porcine bocavirus 1 (PBoV1). Both CnMV and PBoV1 have been found in gastrointestinal infections in their respective hosts, with CnMV responsible for spontaneous abortions in dogs, while PBoV has been associated with encephalomyelitis in piglets. The pathogenicity of the recently identified RBoV is currently unknown. To initiate the characterization of these viruses, their capsids structures were determined by cryo-electron microscopy at resolutions ranging from 2.3 to 2.7 Å. Compared to other parvoviruses, the CnMV, PBoV1, and RBoV capsids showed conserved features, such as the channel at the fivefold symmetry axis. However, major differences were observed at the two- and threefold axes. While CnMV displays prominent threefold protrusions, the same region is more recessed in PBoV1 and RBoV. Furthermore, the typical twofold axis depression of parvoviral capsids is absent in CnMV or very small in PBoV and RBoV. These capsid structures extend the structural portfolio for the Bocaparvovirus genus and will allow future characterization of these pathogens on a molecular level. This is important, as no antivirals or vaccines exist for these viruses.

Keywords: CnMV; PBoV; RBoV; bocavirus; capsid; cryo-EM; parvovirus; pathogen.

Figure 1

Expression and purification of CnMV, RBoV, and PBoV VLPs. (a) An SDS-PAGE of CnMV showing a band at 60 kDa consistent with VP2. To the right, a cryo-EM micrograph of the same sample shows intact capsids of ~25 nm in diameter. (b) Depiction as in (a) for PBoV1 and for (c) RBoV.

Figure 2

Determination of the CnMV, PBoV1, and RBoV capsid structures. (a) Capsid surface density maps of CnMV, PBoV1, and RBoV contoured at a 2 sigma (σ) threshold level. The maps are radially colored (blue to red) according to distance to the capsid center, as indicated by the scale bar in the center. The approximate icosahedral two-, three-, and fivefold axes are indicated. (b) Cross-sectional views of the CnMV, PBoV1, and RBoV density map at a 0.5σ threshold.

Figure 3

The VP structure of CnMV, PBoV1, and RBoV. (a) Amino acid residues modeled for the βD strand are shown inside their density maps at a sigma (σ) threshold of 2.5 (in black). The amino acid residues are labeled and shown as stick representations and colored according to atom type: C = yellow, O = red, N = blue, S = green. These images were generated using UCSF-Chimera [35]. (b) The VP structures are shown as ribbon diagrams inside transparent surface representations. The secondary structure elements (α-helices in red, β-strands in gray), the N- and C-termini, and variable regions (VRs) are labeled.

Figure 4

Sequence and structure comparison within the Bocaparvovirus genus. (a) Superposition of CnMV (purple), PBoV1 (salmon), RBoV (cyan), HBoV1 (blue), and BPV (pink). The variable regions, the N- and C-termini, and the approximate location of the icosahedral two-, three-, and fivefold axes are shown. (b) Amino acid sequence identity comparison of the structurally ordered VP region for the given viruses (top right, in %). The structural similarity is shown in the bottom left corner and was defined as the percentage of aligned Cα atoms of the amino acid chain within 2 Å distance when the capsid structures were superposed.

Figure 5

Location of the VRs on the capsid surface. Capsid surface representations of the bocaviruses with the VRs colored (VR-I = purple, VR-II = blue, VR-III = yellow, VR-IV = red, VR-V = dark gray, VR-VI = pink, VR-VII = cyan, VR-VIII = green, HI-loop = orange, VR-IX = brown). The approximate icosahedral two- (2f), three- (3f), and fivefold (5f) axes are indicated on the CnMV capsid. These images were generated using PyMol [47]. The residue range of the colored VRs is shown in the amino acid sequence alignment below. Amino acid numbering, based on the CnMV sequence, is shown above the alignment. Conserved amino acids are highlighted in gray and the glycines near the N-terminus colored red.

Figure 6

Capsid structures of the genus Bocaparvovirus. Radial dendrogram of selected bocaviruses using their VP2 amino acid sequence, generated at https://ngphylogeny.fr/ (accessed on 12 July 2023). The viruses for which capsid structures were previously determined are colored blue, those that are determined in this study are colored green, and the viruses without a known capsid structure are orange. Capsid surface maps based on the atomic model are shown for CnMV, HBoV2, HBoV1, BPV, PBoV1, and RBoV (ordered clockwise). The maps are radially colored (blue to red) according to distance to the capsid center, as indicated by the scale bar. DBoV: dromedary camel bocavirus, LBoV: rabbit bocavirus, MBoV: mink bocavirus, CslBoV: California sea lion bocavirus, CaBoV: canine bocavirus, FBoV: feline bocavirus.