The Enterovirus 71 A-particle Forms a Gateway to Allow Genome Release: A CryoEM Study of Picornavirus Uncoating

Since its discovery in 1969, enterovirus 71 (EV71) has emerged as a serious worldwide health threat. This human pathogen of the picornavirus family causes hand, foot, and mouth disease, and also has the capacity to invade the central nervous system to cause severe disease and death. Upon binding to a host receptor on the cell surface, the virus begins a two-step uncoating process, first forming an expanded, altered "A-particle", which is primed for genome release. In a second step after endocytosis, an unknown trigger leads to RNA expulsion, generating an intact, empty capsid. Cryo-electron microscopy reconstructions of these two capsid states provide insight into the mechanics of genome release. The EV71 A-particle capsid interacts with the genome near the icosahedral two-fold axis of symmetry, which opens to the external environment via a channel ~10 Å in diameter that is lined with patches of negatively charged residues. After the EV71 genome has been released, the two-fold channel shrinks, though the overall capsid dimensions are conserved. These structural characteristics identify the two-fold channel as the site where a gateway forms and regulates the process of genome release. © 2013 Shingler et al

Lifecycle of a human enterovirus.

<p>Three viral structural proteins (VP0, VP1, and VP3) function as a single structural subunit, the protomer. Five protomers then form a pentamer, twelve of which can self-associate to form a naturally occurring empty capsid (often referred to as procapsid). In pathway one, after the twelve pentamers form an empty procapsid (1A) the genome is inserted (1B). In the second proposed pathway 12 pentamers assemble around a genome to form a provirion (2). In this pathway pentamers that associate in the absence of genome would serve as a reservoir of capsid components in infected cells, and procapsid would be an off-pathway assembly byproduct. The provirion is a short-lived intermediate, as the autocatalytic cleavage of VP0 to form VP2 and VP4 is rapid and efficient. This cleavage results in the native virus, which upon binding to a cellular receptor transitions to an A-particle, which has expelled VP4 and exposed the N-terminus of VP1. An unknown secondary trigger causes the genome to egress, leaving behind an empty capsid shell. Note: The procapsid in this figure is shown in an expanded form, like that of EV71. Other picornavirus procapsids are the same diameter as their native virion (poliovirus and foot and mouth disease virus <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003240#ppat.1003240-Basavappa1" target="_blank">[20]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003240#ppat.1003240-Curry1" target="_blank">[21]</a>).</p

Cryo-electron microscopy image reconstruction data.

<p>Cryo-electron microscopy image reconstruction data.</p

Coulombic surface coloring of the two-fold and five-fold axis.

<p>(A) The EV71 A-particle two-fold axis of symmetry has a high concentration of negatively charged residues (red) with minimal regions of positive charge (blue). This charge distribution will likely serve to aid in the egress of viral RNA by providing a “slippery” surface at the site of genome release. (B) The EV71 five-fold vertex is composed of neutral (white) and positive (blue) charges. In addition to the presence of a density plug at the base of the five-fold, these charges would repel the RNA suggesting the five-fold is not the site of genome expulsion.</p

Cryo-EM reconstructions of the EV71 A-particle and empty capsid.

<p>(A, C) Surface rendered cryo-EM maps of the 6.3 Å EV71 A-particle and the 9.2 Å EV71 empty capsid are shown at a contour level of 1σ colored radially as indicated by the scale bar that depicts the most internal buried density in red gradating to the most external density in dark blue). The five-fold, three-fold, and two-fold axes are labeled. (B, D) Central sections of the EV71 A-particle and empty capsid with density shown in grey. The A-particle contains density corresponding to viral RNA (grey interior), whereas the empty capsid is completely devoid of interior density, indicating the genome has been released. Symmetry axes are labeled and the plug of density that closes the pore through the five-fold vertex is indicated on the central sections of the A-particle and empty capsid.</p

Capsid-RNA interactions in the EV71 A-particle.

<p>Stereogram of the EV71 A-particle showing two columns of protein density extending from the capsid shell to interact with the viral RNA genome. The fitted procapsid crystal structure (3VBU) is depicted as a ribbon diagram with VP0, VP1, and VP3 in green, blue, and red, respectively <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003240#ppat.1003240-Wang1" target="_blank">[30]</a>. The A-particle cryoEM density is depicted as grey mesh. The first ordered N-terminal residue of VP1 in the crystal structure, His73, is shown as cyan spheres and extends into the protein density. The unfilled density beneath His 73 is attributed to the N-terminus of VP1, which is disordered and therefore absent in the procapsid crystal structure, but present in the A-particle.</p

Model for EV71 uncoating.

<p>(A) Binding of a cellular receptor to the capsid of the mature virus (or heating) results in an expanded A-particle. This capsid conformation is characterized by the openings at each two-fold axis of symmetry, the externalization of the N-terminus of the VP1 protein (residues 1–72) from the base of the canyon, and the expulsion of the VP4 protein. (B) If the A-particle forms in proximity to a cellular membrane the VP4 proteins form a channel through which the RNA can enter the host cell cytoplasm. The exposed N-terminii of VP1 proteins anchor the A-particle to the membrane via amphipathic helices.</p

Assessment of the absolute pixel size of the CryoEM maps.

<p>Assessment of the absolute pixel size of the CryoEM maps.</p

EV71 difference map interpretation.

<p>Central sections of calculated difference density maps and close-ups of relevant features are shown to indicate the position of the N-terminus of VP1. (A) The empty capsid density map was subtracted from 9 Å A-particle density map. The difference density (red) is shown superimposed on the A-particle map. The difference density corresponds to the RNA in the particle center, and does not fill the pillars that extend inward from the capsid shell. This can be seen clearly in the close-up of the right upper quadrant of the central section. (B) The calculated procapsid density map was subtracted from the 6.3 Å A-particle density map. The difference density (red) is shown in relation to the A-particle map (grey). The difference density fills the pillars that surround the 5-fold axis and also corresponds to the viral genome in the interior of the particle. The close-up of the upper right quadrant shows that the pillar is mostly filled with difference density (red). (C) The calculated procapsid map was subtracted from the empty capsid density map. The resulting difference density (red) is shown on the empty capsid map (grey). The close-up view shows that the difference density is localized to the pillar (solid arrow head), base of the canyon (unfilled arrow head), and capsid exterior near the 5-fold axis. These same densities are seen in their symmetry related positions with a small noisy density at the tip of the five-fold plug also present.</p