Fig 1.
EV-E assembly pathways and SELEX against EV-E ECs.
(A) Proposed enterovirus capsid assembly pathways. The viral polyprotein is cleaved by the virally-encoded 3CD protease forming protomers that rapidly self-assemble into pentamers, a proportion of which in turn self-assemble into RNA-free ECs in which VP0 subunits remain intact. It has been proposed that pentamers can interact with genomic RNA and assemble directly or enter preformed ECs, both pathways generating a procapsid. Maturation to infectious virion occurs following cleavage of VP0 into VP2 and VP4. These species are distinguishable by centrifugation and their respective sedimentation coefficients are shown. (B) Bernoulli Plot of Sequence Identities of anti-EV-E CP pentamer aptamers to the gRNA (7,414 nts long NC_001859). Each aptamer selected region was assessed for sequence matches to the genome, equivalent to 12 continuous identities, by sliding the sequence across the gRNA 5′ to 3′ incrementing 1 nt at a time. At each nucleotide position fulfilling this requirement a counter was incremented by 1 for each match, resulting in the peaks seen in red. In grey is the equivalent for the unselected starting library. Peaks 7, 9, 21, 24 have matching frequencies ~ 20, 37, 11 and 15 x103, respectively, i.e. are well off the scale of the y-axis. The gene map is shown below the Bernoulli plot. (C) Mfold structure of the SL that can form from the gRNA sequence in Peak #9, the highest peak in (B). (D) Alignment of loop motif regions identified from SELEX peaks. A common RAR motif is found.
Fig 2.
Cryo-EM 3D reconstruction of EV-E virions at 2.2 Å resolution.
(A) Radially colour-coded cryo-EM density map of EV-E capsid shown at 2 σ and viewed along a two-fold axis. One of the pentamers is coloured in orange and the asymmetric unit is coloured following the “classic” colour-code for picornaviruses, VP1 in blue, VP2 in green, VP3 in red and VP4 in yellow. Bar = 50 Å. (B) Atomic model of the asymmetric unit of EV-E shown as ribbon diagrams (top view, left; side view, right) colour-coded as in (A). Symbols and arrows indicate icosahedral symmetry axes. (C) Atomic model of EV-E viral proteins shown as ribbon diagrams and colour-coded as previously. First and last residues at N- and C- termini are indicated. Amino acid side chains for residues involved in contact with gRNA are shown as heteroatom stick diagrams and indicated by dotted circles. Updated atomic coordinates for VP1, VP2 and VP4 are coloured in pink. (D) VP1 pocket factor and (E) VP4 N-terminal domain densities filled by myristic acid molecules shown as stick diagrams and colour-coded as previously, fitted into the 2.2 Å resolution cryo-EM density map shown as grey mesh. Residues are indicated and coloured by heteroatom.
Fig 3.
Contacts between CP and gRNA in EV-E.
(A) Radially colour-coded slab of the cryo-EM unsharpened density map of EV-E capsid shown at 1.6 σ and viewed along a two-fold axis. Dashed circles indicate contacts between CP (dark red) and gRNA (white and blue). Symbols indicate icosahedral symmetry axes. Bar = 50 Å. (B) Atomic model of the front half capsid of EV-E shown as ribbon diagrams, colour-coded as in Fig 2 and viewed along a two-fold axis. Colour-coded dotted circles indicate location of contacts between CP and gRNA on the capsid. (C-H) Detail of the contacts between CP and gRNA indicated in (A, B). The cryo-EM density maps are shown as grey mesh with the density corresponding to RNA facing the bottom part. The atomic model is shown as ribbon diagrams and sticks coloured in orange for the RNA, and sticks colour-coded as previously for the CP. Residues involved in the contact are indicated and coloured by heteroatom. (C) Base stacking contact close to two-fold axis (green dotted circle in A, B) between VP2 W38 and A19-U20 bases of the RNA model generated for the most frequent PS in EV-E (Fig 1B), fitted into a local resolution low-pass filtered density map obtained after symmetry expansion and focused classification on two-fold axes shown at 1.8 σ. (D) Zoom out view of (C) showing fitting of the RNA model into lower resolution density adjacent to the contact. (E) Contact between VP1 R50 and RNA close to two-fold axis (blue dashed circle in A, B) fitted into the same map as in (C) but low-pass filtered to 5 Å resolution shown at 1.3 σ. (F) Contact between VP3 F35 and RNA close to two-fold axis (red dashed circle in A, B) fitted into the icosahedrally averaged map low-pass filtered to 5 Å resolution shown at 1.5 σ. (G) Contact located on the three-fold axis (green dashed circle in A, B) involving VP2 Y9 from three different asymmetric units fitted into the icosahedrally-averaged unsharpened map shown at 1.5 σ. (H) Contact between VP4 G21 and RNA close to five-fold axis (yellow dashed circle in A, B) fitted into the same map as in (G). (I) 3D model of the lowest energy fold for Peak #9 of the Bernoulli Plot obtained by structure prediction in RNA composer shown as in (C, D).
Fig 4.
Reverse genetic analysis of the roles played by contacting CP side chains in virion assembly.
(A) Wild-type and mutant EV-E genome concentrations following transfection, quantified by qPCR of region 6,981–7,081 nts. Orange bars show encapsidated viral RNA concentrations, black lines show total viral RNA concentrations. The dark orange bar labelled virus is EV-E virion infection (using virus concentration equivalent to transfections), Cells refers to cell only control, GNN is EV-E mutant with inactivated replicase. (B) Plaque assays of transfection products. Quantification of three times-passaged EV-E transfection products by plaque assays and qPCR. Following qPCR, the products were run on a gel to confirm the expected length. Lanes on gel were loaded in the order from qPCR graph; only WT showed a band which corresponds with only WT being above detectable limit on qPCR, and only WT producing plaques. (C) Western blot of WT and W38A plasmid transfection lysate probed with anti-VP3 EV-E, stained with Western Blue. (D) Light-scattering of WT (green) and W38A (red) concentrated lysate with poliovirus (PV) ECs as reference (grey, dashed line) fractionated on a TSK 6000 column in PBS. EM of WT and W38A peak fractions, scale bar represents 100 nm. (E) Protein standards run on the same column and conditions as (D) showing a plot of elution points (mL) versus known molecular weight (kDa); vertical dashed line represents the peak elution point of ECs.