Genomic RNA folding mediates assembly of human parechovirus

Figures

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  Fig. 1. Ordered RNA segments in the structures of picornaviruses. Four icosahedrally-symmetric structures for parechovirus virions are currently available. These are HPeV1 (Cryo-EM, 8.5 Å resolution, EMD-1690)7, HPeV1 (X-ray crystallography, 3.1 Å resolution, PDB 4Z92)4, HPeV3 (Cryo-EM, 4.3 Å resolution, EMD-3137)8, and Ljungan virus (Cryo-EM, 4.5 Å resolution, EMD-6395)5. On the top row, an exterior view of each capsid is shown, viewed perpendicular to an icosahedral two-fold axis. On the bottom row, each virus is shown in the same orientation, as a 60 Å thick central section on the left hand side, and the rear half of the capsid on the right hand side. All panels are coloured with an identical radial colour scheme (red: 98 Å, yellow: 111 Å, green: 124 Å, cyan: 137 Å, blue: 151 Å). The diameters of all capsids are very similar, with density ascribed to RNA shown in yellow/red in each case. More RNAs can be seen in the lower resolution HPeV1 EM reconstruction at 8.5 Å, than in the X-ray density at 3.1 Å resolution, suggesting that only a few nucleotides are identical in all 60 positions, but similar stem-loops occupy all positions

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  Fig. 2. Identification of putative PS positions within the HPeV1 Harris genome. a Histogram plot showing alignments of aptamer sequences with Bernoulli scores ≥ 12 to the HPeV1 genome for the enriched SELEX pool (green) and the N40 naïve library (red), base compositions of both pools shown inset. The dashed line, corresponding to the highest peak in the match of the naïve library to the Harris genome, indicates the initial threshold used to identify areas with statistically significant sequence similarities. b Alignment of the results of similar plots for the other 20 complete genome sequences of HPeV1 strain variants are shown with the data from the Harris strain (vertical axis, Harris strain = 1; see Supplementary Table 1 for the Genbank IDs). The positions of statistically significant peaks, i.e. peaks above the level of the highest background peak, within the coding regions of each genome are shown as filled cells in the Table. These were aligned within the polyprotein coding region using Clustal19. Peaks within the Harris strain are termed PSs and are aligned with equivalent sites in the other strains, provided the peak nucleotides lie within 10 nucleotides of the Harris PSs. The…

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  Fig. 3. HPeV1 protein:RNA interactions. A recently published crystal structure by Kalnych et al., PDB: 4Z92, had a new model (PDB: 5MJV) built into the electron density4. a Comparison of the refined crystal structures of the virus with guanosine (left), and adenosine (right) modelled in position 1 of the RNA. Sigma-weighted 2mFo–DFc maps are contoured in grey at 1.0σ. In the right panel, the mFo–DFc difference map is contoured in green at 2.5σ. b Close-up showing a single copy of the viral RNA and its interactions with the surrounding amino acid residues from the viral CPs. a, b Protein and RNA are shown in stick representation with the following colours: oxygen, coral; nitrogen, dark blue; phosphorus, orange. Carbon atoms are coloured by subunit as shown in b, c; additionally, carbon atoms in the RNA base G1 are shown in magenta. Putative hydrogen bonds shown as dashed lines. c View of the viral capsid from inside the virus showing the arrangement of proteins and viral RNA around the icosahedral five-fold. VP1, VP3 and VP0 from one asymmetric unit (denoted VP1a, VP3a and VP0a) are shown in red, green and yellow respectively. VP3 contains a long N-terminal extension that reaches around…

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  Fig. 4. In vitro pentamer binding to the predicted PSs. In vitro analysis of PS: pentamer interactions by MST, HPeV1 pentamers were titrated into fixed amounts of 5ʹ-Alexa 488-labelled short RNAs encompassing PSs 6, 7, 9, 14 and 21 and their variants (labelled m). The binding data were assessed semi-quantitatively (+/−) noting the amplitude of the signal change over the range of pentamer concentrations and where significant binding began to occur. These were compared to a known PS-CP interaction, that of the 19 nt bacteriophage MS2 TR (translational operator) stem-loop and its CP40 (K d~1–4 nM, depending on binding assay used), which was designated as +++++, i.e. low nano-molar affinity. The highest protein concentration samples from these assays were recovered, negatively-stained and examined by electron microscopy (top row shows the PS and the bottom row shows its mutant). Scale bar is 25 nm

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  Fig. 5. Conservation across the HPeV genus. We used the selected HPeV1 library to derive a Bernoulli plot for a representative of the HPeV3 group (GQ183026), which is most distal from the HPeV1 group from the point of view of VP1-VP4 conservation (Supplementary Fig. 6). The number of nucleotide matches of the HPeV3 genome to the selected HPeV1 library is remarkable: with up to 15,000 nucleotide matches for higher peaks and smaller peaks between 1000 and 2000, as opposed to a maximum of 8000 for the higher peaks and 1000–3000 for the lower peaks in HPeV1, indicating that a similar recognition motif is likely to occur also in HPeV3. Consistent with this, sequence fragments ±20 nucleotides around the peak nucleotides can fold into secondary structures that reveal the characteristic GxU motif in the loop of a stem-loop

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  Fig. 6. Cartoon of a plausible virion assembly mechanism. The crystal structure (PDB: 4Z92)4 was used to create a simplified cartoon of the pentameric capsomere. a left, shows a view along the five-fold axis into the virion, i.e. the outer surface. The CP subunits are coloured, yellow for VP0; red for VP1 and various shades of green for the different VP3 subunits. The right hand panel shows the view along the same axis from the centre of the virus, highlighting the extension of the N-terminal arms of the VP3s around the symmetry axis. b shows a similar view with the ordered RNA segments in dark blue. c A cartoon of a hypothesised model of capsid assembly based on known aspects of picornavirus morphogenesis2. Pentamers of the viral CPs are bound by the 2CATPase, associated with a replication factory at the cell membrane via sequence-specific protein-protein contacts to VP1 or VP3. Here they contact newly replicated genomic RNA. The 2CATPase is known to play multiple roles in virion replication and morphogenesis. Its RNA helicase activity would allow it to bias RNA folding to short-range contacts, favouring the appearance of the PSs in HPeV1. These could then associate with their binding…