Fig 1.
Schematic of enterovirus assembly.
Five protomers of VP0, VP3 and VP1 assemble into a pentamer after the P1 precursor is cleaved by 3C or 3CD at the VP0/3 and VP3/1 boundaries. Empty capsids form when 12 pentamers assemble in the absence of genome, these may partially or fully disassemble before reassembly with the RNA genome or be an off-pathway product, with virions directly assembling from pentamers and genome. Provirions exist briefly before genome packaging and the cleavage of VP0 into VP4 and VP2 (red arrow). VP4 is internal, thus is not depicted as a part of the capsid.
Table 1.
Atomic models used as references.
Fig 2.
Molecular characterisation of EVA71 provirions.
A) Samples of WT and E096A mutant EVA71 were recovered from in vitro transcribed RNA electroporated into HeLa cells and separated on 15–45% sucrose gradients. Samples were collected top-down and fractions 5–15 of 17 analysed by western blot using mAb979, which recognises both VP0 and VP2. B) Infectious virus titre of WT virus fractions presented as TCID50/ml, graphed mean ± SEM. C) The presence of genomic RNA in these fractions was assessed by RTqPCR and was quantified relative to a titrated viral sample produced in the same manner. Genome content is presented as percentage genome and is graphed mean ± SEM.
Fig 3.
Surface representation of EVA71 E096A particle classes.
A) Icosahedral 5-3-3 triangle and B) 5-3-2-3 quadrilateral. C) Complete. D) Proximal clipped surface representation of EVA71 E096A particle classes, including details of the clipped surface. Particles coloured by radial distance in Å, indicated in the bottom key. The clipped surface of the particles is coloured grey.
Fig 4.
Surface representation of native EVA71 E096A particles.
Surface rendering of E096A native EC (A & B) and provirion (C & D). Maps are coloured by local resolution in Å, indicated by the map-specific key. Maps are displayed at 1σ. E) Panel describing the clipped surface of the particles in B & D.
Fig 5.
A) Surface rendering of the EVA71 E096A provirion model (left) with VP1 coloured green, VP0 cyan, and VP3 magenta. Position of asymmetric unit indicated in the context of full capsid (middle) and shown separately (right). B) Surface and cartoon representation of the EVA71 E096A protomer viewed from the external surface of the capsid, and rotated 180° along the y-axis, viewing the internal face of the capsid (right) with the approximate location of the internal cleft indicated as a red ellipse. C) Representation of EVA71 E096A provirion and native EC, PV1 native EC (PDB: 1pov), and HRVC native EC (PDB: 5jzg) scissile region including putative catalytic histidine. P1’ residues: E096A; K069, 1pov; N069, 5jzg; M069. Resolved residues upstream of the VP0 scissile boundary coloured yellow for clarity. P1 residue, scissile boundary (*), P2’ residue labelled. Proximal and distal regions of the models have been clipped for clarity.
Fig 6.
Pi- stacking residues and W107.
A) Approximate location of the Y078, R081, W107 interaction indicated in the context of cartoon representation of E096A mutant EVA71 provirion. VP0 residues 12–69 (corresponding to VP4); yellow, residues 70–323; cyan, VP3; magenta, VP1; green. B) Cartoon and stick representation of W107 interacting with Y078 and R081 in EVA71 provirion (E096A), indicated pi-pi and pi-cation interactions with dotted yellow lines with distances shown in Å. C and D) Cartoon and stick representations of W107 interactions in EVA71 E096A provirion (left), and RNA interactions within the mature virion of BEV (6thn) and HRV 14 (7bg6), also displayed as stick Y078 and R081. VP0; cyan, VP3; magenta, VP1; green.
Fig 7.
Phylogeny of VP0 cleaving picornaviruses.
Majority consensus sequences for each ICTV-defined VP0 cleaving picornavirus were generated, and phylogenic analysis performed and a consensus tree built. Sequence alignment covering the region of interest is also shown, with residues predicted to be involved in RNA-dependent VP0 cleavage highlighted. The node which possesses all of these features is boxed in magenta, and key residues conserved within each node are annotated at the relevant node.