Equine rhinitis A virus and its low pH empty particle: clues towards an aphthovirus entry mechanism?

Abstract

Equine rhinitis A virus (ERAV) is closely related to foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae. How picornaviruses introduce their RNA genome into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. It has been thought that the dissociation of the FMDV particle into pentameric subunits at acidic pH is the mechanism for genome release during cell entry, but this raises the problem of how transfer across the endosome membrane of the genome might be facilitated. In contrast, most other picornaviruses form 'altered' particle intermediates (not reported for aphthoviruses) thought to induce membrane pores through which the genome can be transferred. Here we show that ERAV, like FMDV, dissociates into pentamers at mildly acidic pH but demonstrate that dissociation is preceded by the transient formation of empty 80S particles which have released their genome and may represent novel biologically relevant intermediates in the aphthovirus cell entry process. The crystal structures of the native ERAV virus and a low pH form have been determined via highly efficient crystallization and data collection strategies, required due to low virus yields. ERAV is closely similar to FMDV for VP2, VP3 and part of VP4 but VP1 diverges, to give a particle with a pitted surface, as seen in cardioviruses. The low pH particle has internal structure consistent with it representing a pre-dissociation cell entry intermediate. These results suggest a unified mechanism of picornavirus cell entry.

Figure 1. ERAV dissociates at low pH via a transient empty particle.

(A) Schematic depiction of a picornaviral icosahedral capsid (diameter approximately 300 Å) showing the pseudo T = 3 arrangement of 60 copies of each of the viral proteins VP1-4. An individual subunit of the icosahedron (the biological protomer deriving from the uncleaved polyprotein) is coloured conventionally: VP1 blue, VP2 green, VP3 red. VP4 is not shown as it is internal. (B) Sedimentation (from right to left) of radiolabelled virus and products of low pH induced dissociation after exposure to solutions with pH 7.3, 6.5, 5.5 or 4.5 as indicated. Arrows indicate positions on the gradient of virus, empty particles and dissociated pentameric subunits. (C) Sedimentation as (B) after exposure to solutions also containing BSA. (D) Sedimentation of radiolabelled virus and products of low pH induced dissociation after exposure to pH 5.5 for 6, 150 or 750 seconds, as indicated. (E) Empty capsids and RNA sediment separately after exposure to pH 5.5.

Figure 2. Electron density.

(A) Stereo view of the 2Fo-Fc averaged electron density map (green) and coordinates (red) for the N-terminal 17 residues of VP1 in the low pH ERAV particle. (B) Stereo view of the 2Fo-Fc averaged electron density map (green) and coordinates (red) for the N-terminal 17 residues of VP1 in the native ERAV particle. The direction of view is the same as for (A) – note the dramatic rearrangement of this portion of VP1. Both maps are contoured at approximately 0.8 σ.

Figure 3. Structure comparison.

(A) A phylogenetic tree based on a structural alignment of complete protomers (VP1,2,3 & 4) for picornaviruses (RCSB protein data bank code (Berman et al. 2000)): Theiler's virus (1TME), Mengo virus 1 (1MEC), Mengo virus 2 (2MEV) FMDV A10 (1QBE), FMDV O (1BBT), FMDV reduced (1FOD), HRV 14 (4RHV), HRV 16 (1ND2), HRV 1A (1R1A), Swine vesicular disease virus (1OOP), Coxsackie virus 9 (1D4M), Echovirus 1 (1EV1), PV 2 (1EAH). (B) Surface depictions of FMDV (1FOD), ERAV and Mengovirus (2MEV) coloured by radial height (with the same colour scheme for all three particles) to illuminate surface features . (C) 5-fold pores. FMDV, ERAV and PV 1 Mahoney (1HXS) are shown, above as slices through icosahedral 5-fold axes (the axes are vertical), below looking down the pore. In each set of representations the view-point and scale is the same for all three viruses. (D) Capsid porosity measured by the accessibility of viral RNA within the capsid to an RNA binding fluorescent dye at 25°C and after thermal uncoating by incubation at 60°C for ERAV and PV 1 Mahoney.

Figure 4. Protein comparison.

(A) VP1: Overlay , of ribbon depictions from ERAV (magenta), FMDV (1FOD) (green) and Mengovirus (2MEV) (orange) together with the structure derived phylogenetic tree (as Figure 3A) for VP1 proteins with those overlaid highlighted in the same colour. Inset are close-ups of the VP1 pocket in the aphtho and cardioviruses (left) and enteroviruses (poliovirus type 2 Lansing (1EAH) in cyan and, as a further example, swine vesicular disease virus (1OOP) in lime green, right). Both panels include a surface representation in cyan of a sphingosine pocket factor (as bound to swine vesicular disease), although since the site is occluded in aphtho and cardioviruses it is semi-transparent in the left-hand panel. (B) VP2: Overlay and phylogenetic tree (as [A]) for VP2 proteins. Inset is a close-up showing the closer similarity in the VP2 EF loop between ERAV and Mengovirus and the correspondence with the VP1 GH loop (shown in grey with the portion harbouring the integrin binding RGD motif highlighted with a cyan glow) in the reduced FMDV structure (1FOD, [28]). (C) VP3: Overlay and phylogenetic tree for VP3 proteins (viruses, representation and colours as for [A]).

Figure 5. Proposed disassembly intermediate.

(A) Tube depictions coloured as in Figure 1A of the native (left) and low pH (right) protomer structures of ERAV, viewed from the inside of the particle. The regions which change between the structures are highlighted by using a thicker tube. In the native form the VP2 N-terminal hairpin (residues 12–30) is ordered and the N-terminus of VP1 adopts a loop structure stabilizing the VP2/VP3 interface. In the low pH form the VP2 hairpin is disordered and the N-terminus of VP1 has moved to the pentamer interface. His 160, the homologous residue postulated to be involved in the autocatalytic cleavage of VP0 is shown as grey spheres. (B) The variation in pentamer interactions. The representation is similar to (A) and the view is, again, from the inside of the particle.