The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry

Abstract

Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 A X-ray structure of a picobirnavirus--an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two-fold symmetric dimers of a coat protein (CP) with a new 3D-fold. We show that, as many non-enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post-translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120-subunits capsid has evolved animal cell invasion properties.

Figure 1

Structure of the PBV CP. (A) Amino-acid sequence alignment of rabbit and human PBV CP (accession Q9Q1V2 and Q50LE5, respectively) highlighting conserved amino acids. A vertical arrow points to the N-terminal residue of the 55 kD CP found in the particle. Secondary structure elements (SSE) are labelled as α, η and β, for α-helices, 3/10-helices and β-strands, respectively, numbered sequentially from the N terminus and coloured to match the diagrams of the other panels. A labeled circle above the sequence marks residues approaching the icosahedral (I2, I3 and I5) and local (L2) symmetry axes. Small circles below the sequence flag residues involved in L2-related intra-dimer contacts (open circles) and between dimers in the capsid (full coloured circles: blue and red denote single and double (meaning two non-equivalent) contacts in the capsid, respectively. Red triangles below the sequence flag residues involved in the hydrogen-bonding network surrounding the transproteolytic cleavage site. (B) Ribbon diagram of the CP dimer. Shell and Projecting domains are labelled S and P, and the N- and C-termini are indicated. One subunit is coloured by SSE, with all helices in red except for the swapped region (η1-α1, orange) and β-sheets in yellow and green in the S domain, and purple, blue and pink in the P domain. In the blue sheet, dark blue indicates the swapped, N-terminal β1–β2 hairpin. For clarity, the second subunit is shown in grey. (C) Topology diagram. β-strands and α or η helices are indicated by arrows and cylinders, respectively, coloured and numbered as in A, and connected by a black line. A broken line connects elements that are swapped between the two subunits in the dimer. Pale red and blue ovals encompass secondary structure elements of the S and P domains, respectively.

Figure 2

Surface features of the PBV capsid. (A) CP dimer with the subunits in different shades of grey. Top and bottom are views down the L2 axis, from outside and inside the VLP, respectively, with a side view in the middle. The internal curvature of the CP dimer matches roughly the internal radius of the particle. Note the intricate interface between subunits, generated by the N-terminal β1β2 and α1-η1 swapping, as indicated. The location of the N-terminus marks the site of the transproteolytic cleavage. (B) Surface of the VLP. cEM 3D-reconstruction showing the presence of 60 dimeric protrusions formed by domain P. The contour level was chosen to accommodate the volume of the CP dimer in the icosahedral asymmetric unit. A few symmetry axes are labelled for orientation. Top and bottom panels are seen down the I3 and I5 axes, respectively, slightly miss-oriented in order to help visualize certain features, like the cleft at the I2 axes, the I5 depression and the flat I3 region. Some of the symmetry axes are drawn, with standard symbols for 2-, 3- and 5-fod axes (empty ellipse, triangle and pentagon, respectively) and labelled. Bar: 100 Å.

Figure 3

Parallel dimers define the outline of a rhombus-shaped tile on the icosahedral particle. (A) Two CP dimers related by an I2 axis of the particle are displayed as ribbons, with the S and P domains coloured blue and red, respectively. The nearest icosahedral and local symmetry axes are drawn in projection and labelled. The left panel is projected down the I2 axis. The L2 axes make an angle of about 11.5 degrees with the I2 axis and do not intersect each other, but lie in parallel planes perpendicular to the paper; small arrows by the L2 labels indicate the direction and length (6.2 Å) of the translation needed to bring these planes into coincidence with a parallel vertical plane containing the I2 axis. The right panel shows an orthogonal view rotated about a vertical axis. Full-line arcs indicate the inner and outer radii of the particle (labelled), highlighting the curvature of the diamond tile. The L2 axes cross the vertical plane containing the I2 axis (which is in the plane of the paper) along a vertical line, at a distance of 14.5 Å from each other, above and below the I2 axis. This vertical line intersects the I2 axis at a radius of about 35 Å (broken line arc), indicating a sharper local curvature at the I2 contact than that of the overall particle. The particle centre is indicated by O, where I5, I3 and I2 axes intersect. Residues Asn 157 and Asn 221 are displayed as yellow and red spheres and labelled, as well as helices α5 and α6 mentioned in the text. C-ter labels the last four amino acids of chain A (587–590, disordered in chain B) running along the I5 axis. Bar: 100 Å. (B). Triacontahedral design of the PBV particle, each of the 30 rhombic tiles is coloured differently. The red tile is oriented as the one displayed in the top panel. (C) Convergence with the icosahedral organization with the flavivirus particle, where each of the 30 tiles (coloured as in B) is composed of 3 parallel dimers of the envelope protein. Coordinates obtained from PDB entry 1THD. Panels B and C are at the same scale. Bar: 500 Å.

Figure 4

View of the PBV particle down an I5 axis. The individual subunits are coloured according to secondary structure elements: α-helices blue, β-sheets red and random coil orange/light brown. Note the I5 loop with the conserved 157-NSG-159 segment at the centre, forming a diaphragm-like structure at the I5 axis. Bar: 100 Å

Figure 5

Comparison with L-A virus (PDB entry 1M1C) and the BTV inner layer (PDB entry 2 BTV). The particles are displayed in molecular surface representations viewed down an I2 axis, with the two independent subunits coloured blue (chain A, by the I5 axes) and grey (chain B, by the I3 axes). For clarity, a yellow contour outlines two I2-related CP dimers in the PBV particle (left panel). Red full symbols indicate icosahedral axes (triangle for I3 and ellipse for I2). The L2 axes (or distorted L2 axes in the case of L-A virus and BTV, see Supplementary Table S1) are shown by open red ellipses. One of the 12 decameric CP caps is labelled A_n and B_n, n=1 to 5, with the 5 B subunits in light grey. Note the very large interface of each B subunit with its two nearest A neighbors (A₅ and A₁ for B₁, etc). Subunits labelled A'_n and B'_n belong to an I2-related decameric cap with the B subunits coloured intermediate grey; a third cap to the right, related by the labelled I3 axis, is drawn with the B subunits coloured dark grey (not labelled). In contrast to L-A virus and BTV, the PBV dimer mixes subunits from two adjacent decameric caps (AB' and BA'), indicating that the assembly pathway is necessarily different. All three panels are drawn at the same scale. Bar: 500 Å.