Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis

Abstract

The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in complex with human monoclonal antibody Fabs demonstrates the expected picornavirus capsid structure with three distinct features. First, 25% of the HPeV3 RNA genome in 60 sites is highly ordered as confirmed by asymmetric reconstruction, and interacts with conserved regions of the capsid proteins VP1 and VP3. Second, the VP0 N terminus stabilizes the capsid inner surface, in contrast to other picornaviruses where on expulsion as VP4, it forms an RNA translocation channel. Last, VP1's hydrophobic pocket, the binding site for the antipicornaviral drug, pleconaril, is blocked and thus inappropriate for antiviral development. Together, these results suggest a direction for development of neutralizing antibodies, antiviral drugs based on targeting the RNA-protein interactions and dissection of virus assembly on the basis of RNA nucleation.

Figure 1. HPeV3 structure.

(a) Radially coloured isosurface representation down a twofold axis of symmetry of HPeV3 at 4.3 Å resolution shown at 3σ above the mean threshold. The arrow indicates the canyon. Colour key shows the radial colouring from the centre of the virus in nm. (b) Representative fit of VP0 atomic model to electron density (mesh). (c) A zoomed-in view of the capsid model showing the positions of VP0 (yellow), VP1 (red) and VP3 (green) in a T=1 (pseudo T=3) arrangement. The symmetry axes are marked in blue (fivefold pentagon, threefold triangle, twofold ellipse). The capsid is made from 12 pentamers of VP0, VP1 and VP3. Some of the proteins in neighbouring pentamers are marked (VP0, C1-C16; VP1, A1-A16; VP3, B1-B16). (d) A stabilizing network of VP0 N-terminal arms traverses the inner side of the capsid. The path of one N terminus is highlighted in yellow (C1) from pentamer 1 (pink) travelling via VP3 (gold) of pentamer 2 (gold) to interact with the N terminus of C16 from pentamer 3. These VP0 N termini obstruct the pore at the twofold symmetry axis between pentamer 2 and pentamer 4. (e) Unusual position of HPeV3 VP0 N terminus compared to other picornavirus is shown by superposition of VP0 from HPeV3 (yellow), with poliovirus (1pov; orange), EV71 (3vbu, magenta) and HAV (4qpg, blue). In addition, the locations of VP4 (cyan) and VP2 (white) of EV71 (3vbf) in comparison to HPeV3 VP0 are also shown. N termini for all the superimposed proteins are marked with asterisks in e). (f) HPeV3 VP1 β-barrel region is shown superimposed on a pocket factor containing EV-D68 VP1 (4wm7; VP1, blue; pocket factor, grey). The HPeV3 hydrophobic pocket is blocked by residues Y133, F163 and Y164.

Figure 2. Ordered RNA–protein interactions.

(a) Central cross-section of the unfiltered HPeV3 icosahedrally-symmetric reconstruction with three symmetry axes marked. The red circle in (a) and (c) indicates one of the regions where the capsid proteins interact with finger-like RNA densities. (b) Central cross-section of the unfiltered HPeV3 asymmetric reconstruction. (c) Enlargement of the VP3 (green) and VP1 (red) atomic model in intimate contact with an RNA model (magenta, R1) within its asymmetric unit. The VP3N terminus also interacts with a neighbouring RNA molecule within the pentamer. The fit of one of the RNA models in the RNA EM density (transparent isosurface) is shown. The RNA interacting regions in VP1 and VP3 are coloured black. (d) The icosahedrally-symmetric copies of the fitted-RNA model from c shown in the HPeV3 asymmetric reconstruction's RNA density (yellow transparent surface shown at 2σ above the mean threshold). (e) VP1 and VP3 interaction with the RNA is shown in the context of the inner surface of a pentamer. The N terminus of VP3 (B1) and regions of VP1 (A1) interact with the RNA (R1) within its asymmetric unit and also with an RNA (R3) from the next-but-one asymmetric unit within a pentamer. The proteins are marked as in Fig. 1c. The RNA models are marked R1-R5 for their respective asymmetric units as in Fig. 1c). (f) The inner surface of a pentamer of the HPeV3 model shown as an electrostatic potential surface with the conserved RNA motif (magenta) shown in ribbon. The scale for the charge distribution is also shown. The RNA interaction with the capsid protein does not appear to be driven by electrostatics as the interacting region on the capsid proteins appear to be a mix of positive (blue), negative (red) and neutral charges (white).

Figure 3. Structure-based sequence alignment of human parechoviruses.

Multiple sequence alignment of HPeV1-8 amino acid sequences of capsid proteins VP0, VP3 and VP1 using protein BLAST is shown. The secondary structure elements from the atomic model of HPeV3 152037 are shown above the alignment as α-helices (spirals), β-sheets (arrows), disordered regions (dashed lines). Sequence annotations on the left correspond to the virus genotype/GenBank IDs. Sequence identity among all the aligned human parechoviruses (blue highlight), RNA binding sites (black boxes) and conformational epitope for mAb AT12-015 (red lines) are indicated on the HPeV3 isolate 152037 protein sequences. Disordered regions of the structural proteins were truncated from the atomic HPeV3 model. The disordered VP1 C terminus is exposed on the outside of the capsid and was identified in the footprint of Fab AT12-015 by comparison to other picornavirus structures. The footprint incorporates four amino acid differences between HPeV3 152037 and A308/99 in the variable surface loop VP3 βChA and the C terminus of VP1. The amino acid sequence identity of 19 P1 polyproteins from all HPeV3 isolates available in GenBank is 99%.

Figure 4. HPeV3–Fab structure.

(a) Radially coloured isosurface representation of HPeV3–Fab AT12-015 complex at 15 Å resolution shown at 1.5σ above the mean threshold. The Fab molecules (red) bind around the canyon region. (b) HPeV3 roadmap. The Fab footprint (red contour) is mapped to VP3 (green) and VP1 (red).