Cryo-electron microscopy and image classification reveal the existence and structure of the coxsackievirus A6 virion

ARTICLE https://doi.org/10.1038/s42003-022-03863-2 OPEN Cryo-electron microscopy and image classification reveal the existence and structure of the coxsackievirus A6 virion 1234567890():,; Carina R. Büttner 1, Radovan Spurný1, Tibor Füzik 1 & Pavel Plevka 1✉ Coxsackievirus A6 (CV-A6) has recently overtaken enterovirus A71 and CV-A16 as the primary causative agent of hand, foot, and mouth disease worldwide. Virions of CV-A6 were not identified in previous structural studies, and it was speculated that the virus is unique among enteroviruses in using altered particles with expanded capsids to infect cells. In contrast, the virions of other enteroviruses are required for infection. Here we used cryoelectron microscopy (cryo-EM) to determine the structures of the CV-A6 virion, altered particle, and empty capsid. We show that the CV-A6 virion has features characteristic of virions of other enteroviruses, including a compact capsid, VP4 attached to the inner capsid surface, and fatty acid-like molecules occupying the hydrophobic pockets in VP1 subunits. Furthermore, we found that in a purified sample of CV-A6, the ratio of infectious units to virions is 1 to 500. Therefore, it is likely that virions of CV-A6 initiate infection, like those of other enteroviruses. Our results provide evidence that future vaccines against CV-A6 should target its virions instead of the antigenically distinct altered particles. Furthermore, the structure of the virion provides the basis for the rational development of capsid-binding inhibitors that block the genome release of CV-A6. 1 Central European Institute of Technology - Masaryk University, Structural Biology, Structural Virology, Kamenice 5, 62500 Brno, Czech Republic. ✉email: COMMUNICATIONS BIOLOGY | (2022)5:898 | https://doi.org/10.1038/s42003-022-03863-2 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-03863-2 E nteroviruses are a globally distributed and ubiquitous threat to humans and livestock, causing infections of the respiratory and gastrointestinal tracts, skin, and central nervous system1–3. In recent years, Coxsackievirus A6 (CV-A6) has overtaken enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) as the major causative agent of hand, foot and mouth disease (HFMD) in young children4. The virus is also emerging as a frequent causative agent of HFMD in adolescents5. Unlike the classical HFMD viruses EV-A71 and CV-A16, CV-A6 is associated with atypical HFMD, which manifests as systemic large vesicular eruptions and onychomadesis or orchiepididimitis, but can also cause aseptic meningitis or encephalomeningitis4,6–9. Common in Southeast Asia, where it has been the principal cause of HFMD since the early 2010s10–14, CV-A6 has since spread globally15–21. Strains of CV-A6 that cause severe clinical symptoms in adults have been identified19,22. Available HFMD treatments and vaccines are predominantly directed against EV-A71 and CV-A16, whereas no vaccine exists to counter CV-A6. Picornaviruses, including viruses from the genus Enterovirus such as CV-A6, CV-A16 and EV-A71, are small, non-enveloped icosahedral viruses with single-stranded positive sense RNA genomes (reviewed in ref. 23). To initiate infection, enterovirus particles progress through a set of distinct functional intermediates: the infectious native virion expands into a destabilised altered or A-particle, in which the internal capsid protein VP4 has been displaced24. The genome is released through a capsid opening, leaving the empty capsid as the non-infectious end product25–28. Although high-resolution structures of the EV-A71 and CV-A16 virions are available29–31, the structural characterisation of CV-A6 is limited to altered and empty particles and virus-like particles32,33. The absence of virions in CV-A6 preparations led Xu et al. (2017) to speculate that the altered particle is the sole infectious species of CV-A6 and an exception to the canonical enterovirus infection pathway32. Here we report the structure of the previously unrecognised CV-A6 virion. We determined cryo-EM structures of the compact CV-A6 virion, the expanded altered particle, and the empty particle to a better than 3-Å resolution. We used a fluorescencebased infection assay to show that CV-A6 has an infectious unitto-particle ratio of 1: 500. Our findings reestablish the canonical steps of the enterovirus infection process for CV-A6 and enable the rational design of capsid-binding therapeutics against CV-A6. Results and discussion CV-A6 virion, altered, and empty particles. The sample of purified CV-A6 contained a mixture of genome-containing and empty particles (Fig. 1 and Supplementary Fig. 1a). The combination of two-dimensional (2D) and 3D classification identified 1769 virions (1.2%) in 131,286 images of altered particles with expanded capsids (91.4%) and 10,613 empty particles (7.4%) (Supplementary Fig. 2). The structure of the CV-A6 virion was determined to a resolution of 2.68 Å, whereas those of the altered and empty particles reached resolutions of 2.50 and 2.82 Å, respectively (Fig. 1, Table 1 and Supplementary Fig. 2d–f). Overall, the CV-A6 virion exhibits surface features typical for the virions of enteroviruses, including the prominent star-shaped plateau (mesa) around each fivefold symmetry axis, a pronounced depression (canyon) encircling the mesa, and three-bladed propeller shapes around the threefold symmetry axes (Fig. 1). The virion is 3.2% smaller than the altered and empty particles, a similar change in size to that observed for other enteroviruses such as EV-A71, CV-A16, CV-B3, and poliovirus 130,31,34,35. The virion has less pronounced surface features (Fig. 1a–c). Unlike the altered and empty particles, the CV-A6 virion does not contain openings located on the twofold symmetry axes of its capsid (Fig. 1a). The smaller radius and the absence of open channels in the compact structure of the CV-A6 virion provide evidence that it represents the genuine native conformation of a mature enterovirus. The structures of the altered and empty CV-A6 particles reported here are similar to those of the altered particle (Protein Data Bank (PDB) entry 5XS4) and procapsid (5XS5) reported previously32. The corresponding maps can be superimposed with correlation coefficients of 0.94 and 0.95, and the protomer structures with an overall root mean square deviation (r.m.s.d.) of 0.73 and 0.75 Å, respectively, matching 612 of 623 and 594 of 610 Cα atoms available for the comparisons (Supplementary Fig. 3 and Supplementary Table 1). The similarity is expected for closely related virus strains Gdula, used in this study, and TW-200700141 which share 94, 96, and 96% sequence identity for VP1, VP2, and VP3, respectively (Supplementary Fig. 4). Structure of the CV-A6 virion. The capsid of the CV-A6 virion is built from 60 protomers composed of subunits VP1, VP2, VP3, and VP4 (Fig. 2a, b). The major capsid proteins VP1, VP2, and VP3 are arrange (...truncated)