Hepatitis A virus and the origins of picornaviruses

Abstract

Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2 'domain swap' characteristic of insect picorna-like viruses, and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between 'modern' picornaviruses and the more 'primitive' precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.

Extended Data Figure 1. HAV purification and characterization

a, Zonal ultracentrifugation of a 15 to 45% (w/v) sucrose density gradient at 103614 g for 3.5 h was used to purify HAV, as described in Methods. Two predominant particle types were separated; one was located at ~27% sucrose, the other at ~32% sucrose. The absorbance ratios (λ260/λ280) were 0.76 for the top band and 1.66 for the bottom band indicating that the former contained mainly empty particles without the RNA genome and the latter mainly full particles with the RNA genome. The top band was much broader than the bottom one. Three and two fractions were collected around the top and the bottom bands, respectively, for further purification. b, SDS-PAGE for protein composition analysis using A NuPAGE 4–12% Bis-Tris Gel (Invitrogen). Each lane was loaded with 5-10 μg sample (lane 1, full particles; lane 2, empty particles; lane 3, markers). The calculated molecular weights of VP0, VP1, VP2, VP3 and VP4 were 27.26 kD, 30.73 kD, 24.80 kD, 27.86 kD and 2.50 kD, respectively. The results appear to show incomplete cleavage of VP0 in the full particles and no VP0 cleavage in the empty particles. c,d, Sedimentation velocity experiments were performed on a Beckman XL-I analytical ultracentrifuge at 20 °C. Samples were loaded into a conventional double-sector quartz cell and mounted in a Beckman four-hole An-60 Ti rotor. Data were collected at 15,000 rpm at a wavelength of 287 nm. Interference sedimentation coefficient distributions were calculated from the sedimentation velocity data using SEDFIT. The full particle has a sedimentation coefficient of 144S and the empty particle 82S. e,f, Negative stain electron microscopy of HAV particles. Particles from the top band in panel a, shown in panel f, suggest that light sedimentation fractions were mainly composed of empty particles. Note that some particles appear to have external features. Panel e shows the heavy particles, which appear to contain viral RNA.

Extended Data Figure 2. HAV capsid protein structure

a,c, Stereo diagrams showing the structures of the capsid proteins VP1, VP2 and VP3, respectively. The Cα backbone is shown as a thin line, the N and C termini are labeled, every 10^th residue is marked with a small sphere and every 20^th is numbered. d, VP1 is initially produced with an 8 kD C-terminal extension (known as PX) unique to HAV among picornaviruses. PX is cleaved from the full particle by an unknown host protease. According to the data shown in Extended Data Figure 1 and the electron density maps, the particles we have analysed do not contain PX, but if it were present we would expect its course to start at the purple positions (C-termini of VP1) on the surface of the virus.

Extended Data Figure 3. Antigenicity of Full and Empty Particles

The reactivity of HAV full and empty particles against a panel of 6 purified HAV neutralizing mAbs was measured by ELISA. The bar charts represent the average OD450 reading for the six mAbs at each dilution with the standard deviation shown as an error bar.

Extended Data Figure 4. PaSTRy assays

To characterize the stability of HAV full and empty particles compared to CrPv across the pH range from 2.0 to 10.0 differential scanning fluorimetry assays were performed with dyes SYTO9 (to detect RNA exposure) and SYPRO RED (to detect protein melting). a, The raw fluorescence traces of HAV full particles incubated with SYTO9. b, The raw fluorescence traces of HAV full particles incubated with SYPRO red. c, The raw fluorescence traces of HAV empty particles incubated with SYPRO red. d, The raw fluorescence traces of CrPV full particles incubated with SYTO9 across the same pH range. The colour scheme is dark red (pH 2.0), red (pH 3.0), orange (pH 4.0), yellow (pH 5.0), green (pH 6.0), sky blue (pH 7.0), blue (pH 8.0), dark blue (pH 9.0) and purple (pH 10.0). Since the SYTO9 dye didn’t function well below pH 4.0 the fluorescence traces for pH 2.0 and pH 3.0 are omitted. These results indicate that HAV full virions are most stable at pH 5.0 and RNA genome release occurs at about 76 °C and protein melting at 77 °C, i.e. there is no notable transition between RNA release and particle loss. HAV empty particles show a similar trend but appear to withstand temperatures up to 81°C in low pH buffer. In contrast CrPV is most stable at pH 4.0 and RNA genome release occurs at about 54 °C.

Extended Data Figure 5. Interactions between α1 helices of VP2 at the icosahedral 2-fold

a, The close-packing of the two helices is shown in particular the packing of Tyr 100 against the adjacent helix. The contact area between these helices and the surface complementarity suggests a well fitting interface for HAV (contact area 83 Ų, surface complimentarity 0.755, EV71: 111/0.550, Polio-1: 124/0.785, CrPV: 72/0.707, FMDV: 50/0.593). The interface between protomers forming the pentameric units is similar for all these viruses (HAV: 4267 Ų, Polio-1: 4369 Ų, CrPV: 4647 Ų, EV71: 4131 Ų, FMDV-A22: 3432 Ų). b, Helical wheel diagrams for helix α1 of several picornaviruses showing the unusually small side chains at the helix interface in HAV.

Extended Data Figure 6. Antigenicity and YPX₃L ALIX-interacting motifs

a, Previously indicated antigenic sites of HAV are mapped onto the structure (pink spheres). Late domain YPX₃L motifs are shown as orange spheres. b,d, Surface maps of HAV generated using RIVEM such that area for each residue as drawn corresponds to its accessible area and the coloration is according to the radius from the virus centre (Blue deepest, red most exposed). On these the antigenic sites of HAV are depicted in purple. b, Previously reported antigenic sites; c, predicted sites; d, new sites determined by peptide mapping (indigo) together with previously identified sites (purple).

Extended Data Figure 7. mAb neutralization assays and peptide epitope mapping

a, In vitro neutralization assays of monoclonal antibodies against HAV (TZ84). b, Peptides used for epitope mapping. c, Reactivity of the neutralizing mAb #11 against synthetic peptides measured by peptide-ELISA.

Figure 1. Overall structure

a, HAV accessible surface (VP1: blue, VP2: green, VP3: red for all panels). Black lines: particle facets, white outline: biological protomer. b, Surface of the biological protomer of HAV and poliovirus. Loops forming the canyon walls in poliovirus are drawn thicker. c, HAV electrostatic surface (using APBS in PyMOL). Red negative, blue positive, white neutral, sulphate ions yellow. d, HAV viewed from inside. Blue positive |F_o-F_c| electron density calculated taking the correctly positioned empty HAV from the full HAV shows that VP1 2-28 (darker density) and VP2 5-17 are better defined in the full particle.

Figure 2. Structure features

a HAV and b EV71 coloured as 1a, light blue mesh: pocket-volume calculated with PyMOL. Magenta: EV71 pocket factor. c, Pocket close-up, left strand C, right H, for: HRV14 (yellow), FMDV (blue), EV71 (orange, pocket factor magenta) and HAV (green, note occlusion of pocket entrance). Met224 occludes part of the empty HRV14 pocket. A and B: bulky side-chains occlude the HAV and FMDV pockets (A: Leu-HAV, Tyr-FMDV, B: Phe-HAV and Tyr-FMDV). d, e, Biological protomers of CrPV and FMDV respectively superimposed on HAV. The star marks the VP2 N-terminus that folds differently in FMDV (switch at residue 53 is indicated). HAV coloured as in a (CrPV and FMDV grey). f, HAV compared with polio virus type 1 (1HXS) in g. The pentamer interface runs horizontally across the centre with the perpendicular 2-fold axis roughly central. Surface drawn for upper pentamer. 2-fold related β-sheets are at A and B. VP1 blue and indigo, VP2 green and lime-green, VP3 red. VP4 omitted for clarity. Upper pentamer chains drawn thicker.

Figure 3. Stability

a, Summary of thermal shift assays for HAV in the pH range from 2 to 10. Purple and green bars represent RNA release and protein-melting temperatures respectively for full HAV, blue bars show protein melting for the empty particle. RNA release is not detected at low pH due to quenching of the SYTO9 dye. Extended Data Fig. 4 shows raw fluorescence traces. b, The separation of VP2 α-helices at the icosahedral 2-fold of HAV (green, 3.8Å), CrPV (blue, 5.3Å), EV71 (cyan, 7.3Å), polio (grey, 7.6Å ), FMDV (orange, 8.2Å) and 80S-like EV71 (red, 12.2Å).

Figure 4. Phylogeny

a, Structure-based phylogenetic tree of representative picornaviruses and cripaviruses, 3VBF (EV71), 1BEV (bovine enterovirus), 4HRV (human rhinovirus14), 1HXS (poliovirus type1), 1COV (coxsackievirus B3), 1TME (Theilers virus), 3MEV (mengo virus), 3CJI (Seneca valley virus), 1ZBA (FMDV A10), 2WFF (equine rhinitis A virus), 3NAP (triatoma virus) and 1B35 (CrPV). Evolutionary distance is derived from the number of unmatched residues and the deviation in matched residues. Residues corresponding to the HAV VP2 switch region (1-53) are excluded (including them does not affect the result). b, Superimposition of HAV VP1 (blue), VP2 (green) and VP3 (red), note the similar N-terminal extensions.