Fig 1.
The 3.1-Å structure of wtMNV solved by cryo-EM.
(a) Isosurface representation of the 3.1 Å wtMNV density map, shown at 1 σ and coloured according to the radial colouring scheme shown (Å). The 5-fold (hexagon), 3-fold (triangle), and 2-fold (oval) icosahedral axes are indicated. (b,c,d) Enlarged views centred on the icosahedral 2-fold axes of (b) our apo wtMNV reconstruction, (c) the recently published cryo-EM reconstruction of apo wtMNV from Sherman and colleagues [10], or (d) the reconstruction of wtMNV in complex with GCDCA reported by Sherman and colleagues [10]. All maps were low-pass filtered to 4.0 Å resolution and coloured according to the radial colouring scheme shown (Å). The angle of rotation for AB- or CC-type P domain dimers compared to the wtMNV reconstruction shown in (b) are given. Central sections through each reconstruction are also shown. (f,g) Side views of example class averages from focussed classification of (f) AB-type or (g) CC-type P domain dimers, with the atomic coordinates for wtMNV VP1 P domain dimers rigid-body fitted in each case. (h) Overlaid atomic coordinates for different quasiequivalent copies of MNV VP1, shown by the colouring scheme. The most N-terminal residue modelled for each quasiconformer is indicated by NA (D20), NB (S17), or NC (V30). (i) Atomic coordinates for N-terminal regions of adjacent A-type VP1 molecules, with polar contacts shown with blue dashed lines. cryo-EM, cryo-electron microscopy; GCDCA, glychochenodeoxycholic acid; MNV, murine norovirus; P domain, protruding domain; S domain, shell domain; VP, viral protein; wt, wild type.
Fig 2.
Thermal inactivation of wtMNV.
(a) Samples of wtMNV were incubated at a range of temperatures up to 70°C on a 30-second constant temperature ramp before being immediately cooled on ice. Titres were determined by TCID50 assay on RAW264.7 cells (n = 2 ± SEM). (b) wtMNV was purified by sucrose density gradient, dialysed into PBS, and used for PaSTRy thermal stability assays using the nucleic acid dye SYTO-9 (green) and the protein dye SYPRO-Orange (orange) on a 30-second constant temperature ramp (n = 3 ± SEM). (c) Samples of MNV were heated to the indicated temperatures, treated with RNase A, and then titrated by TCID50 assay on RAW264.7 cells (n = 2 ± SEM) or used to extract total RNA. The extracted RNA was transfected into BHK cells (which only permit a single round of replication), and resultant virus was harvested and titrated by TCID50 assay on RAW264.7 cells (n = 2 ± SEM). Numerical data for Fig 2 are provided in S1 Data. BHK, baby hamster kidney; MNV, murine norovirus; PaSTRy, Particle Stability Thermal Release; PBS, phosphate-buffered saline; TCID50, median tissue culture infectious dose; wt, wild type.
Fig 3.
hiMNV has weaker P domain density than wtMNV.
(a) Isosurface representations of the 3.1-Å wtMNV reconstruction (wt) and the 2.9-Å hiMNV reconstruction (hi), contoured to different thresholds (1 σ–4 σ). (b) Example focussed classes from focussed classification of wtMNV and hiMNV CC-type P domain dimers, shown at 2 σ. The sites of contact between C-type and A-type P domains, used to determine ‘noncontacting’ status, are indicated by white arrowheads. All focussed classes are shown in S4 Fig (wtMNV) and S6 Fig (hiMNV). hiMNV, heat-inactivated MNV; MNV, murine norovirus; P domain, protruding domain; wt, wild type.
Fig 4.
Selection and biochemical characterisation of hsMNV.
(a) Samples of MNV were heated at 52°C for 30 min before cooling to 4°C. The surviving pool of viruses was subsequently passaged for 48 hours at 37°C on RAW264.7 cells. Prior to selection (‘wt’) and at each passage, the virus titre was determined by TCID50 on RAW264.7 cells (n = 2 ± SEM). Consecutive cycles of selection were performed. (b) The pool of virus heated at 52°C between passages (termed MNV52) and wtMNV were heated to a range of temperatures between 37°C and 70°C and virus titre determined by TCID50 assay on RAW264.7 cells (n = 4 ± SEM). (c) Virus samples were purified by sucrose density gradient, dialysed into PBS, and used for PaSTRy thermal stability assays using the nucleic acid dye SYTO-9. (d) The antigenicity of wtMNV or hsMNV was determined by ELISA with anti-VP1 antibodies, 2D3 and 4F9, after incubation at the indicated temperature (n = 2 ± SD). Numerical data for Fig 4 are provided in S2 Data. hsMNV, heat-stable MNV; MNV, murine norovirus; PaSTRy, Particle Stability Thermal Release; PBS, phosphate-buffered saline; TCID50, median tissue culture infectious dose; VP, viral protein; wt, wild type.
Fig 5.
hsMNV has ‘twisted’ P domain dimers relative to wtMNV.
(a) Overlaid isosurface representations of wtMNV (grey) and hsMNV (orange) centred on the icosahedral 2-fold axis, shown at 1 σ after low-pass filtering both maps to the same resolution (4.0 Å). The back plane is clipped to remove the S domains. (b) Atomic coordinates for AB-type VP1 fitted into wtMNV (grey) or hsMNV (orange) density maps, centred on the AB dimer–CC dimer interface highlighted by the white arrowhead in (a). S domains are shown in green. The mutated residue is shown as magenta (wtMNV, L412) or dark purple (hsMNV, L412Q) spheres. Angles of rotation in 2 axes are indicated. (c) A ‘noncontacting’ class resulting from focussed classification of hsMNV CC-type P domains, shown at 2 σ. hsMNV, heat-stable MNV; MNV, murine norovirus; P domain, protruding domain; S domain, shell domain; VP, viral protein; wt, wild type.