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 cryo-electron 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.

Central European Institute of Technology Masaryk University Structural Biology Structural Virology Kamenice 5 62500 Brno Czech Republic

Zobrazit více v PubMed

Tapparel C, Siegrist F, Petty TJ, Kaiser L. Picornavirus and enterovirus diversity with associated human diseases. Infect. Genet. Evol. 2013;14:282–293. doi: 10.1016/j.meegid.2012.10.016. PubMed DOI

Rhoades RE, Tabor-Godwin JM, Tsueng G, Feuer R. Enterovirus infections of the central nervous system. Virology. 2011;411:288–305. doi: 10.1016/j.virol.2010.12.014. PubMed DOI PMC

Hughes, L. E. & Ryan, M. D. in Encyclopedia of Virology (Third Edition) (eds Mahy, B. W. J. & Van Regenmortel, M. H. V.) (Academic Press, 2008).

Bian L, et al. Coxsackievirus A6: a new emerging pathogen causing hand, foot and mouth disease outbreaks worldwide. Expert Rev. Anti Infect. Ther. 2015;13:1061–1071. doi: 10.1586/14787210.2015.1058156. PubMed DOI

Ang LW, et al. Seroepidemiology of coxsackievirus A6, coxsackievirus A16, and enterovirus 71 infections among children and adolescents in Singapore, 2008-2010. PLoS ONE. 2015;10:e0127999. doi: 10.1371/journal.pone.0127999. PubMed DOI PMC

Österback R, et al. Coxsackievirus A6 and hand, foot, and mouth disease, Finland. Emerg. Infect. Dis. 2009;15:1485–1488. doi: 10.3201/eid1509.090438. PubMed DOI PMC

Fujimoto T, et al. Hand, foot, and mouth disease caused by coxsackievirus A6, Japan, 2011. Emerg. Infect. Dis. 2012;18:337–339. doi: 10.3201/eid1802.111147. PubMed DOI PMC

Fujimoto, T. [Hand-foot-and-mouth disease, aseptic meningitis, and encephalitis caused by enterovirus]. Brain Nerve70, 121–131 (2018). PubMed

Li Y, et al. Emerging enteroviruses causing hand, foot and mouth disease, China, 2010-2016. Emerg. Infect. Dis. 2018;24:1902–1906. doi: 10.3201/eid2410.171953. PubMed DOI PMC

Gao L, et al. Spectrum of enterovirus serotypes causing uncomplicated hand, foot, and mouth disease and enteroviral diagnostic yield of different clinical samples. Clin. Infect. Dis. 2018;67:1729–1735. doi: 10.1093/cid/ciy341. PubMed DOI

He S, et al. An emerging and expanding clade accounts for the persistent outbreak of coxsackievirus A6-associated hand, foot, and mouth disease in China since 2013. Virology. 2018;518:328–334. doi: 10.1016/j.virol.2018.03.012. PubMed DOI

Anh NT, et al. Emerging coxsackievirus A6 causing hand, foot and mouth disease, Vietnam. Emerg. Infect. Dis. 2018;24:654–662. doi: 10.3201/eid2404.171298. PubMed DOI PMC

Puenpa J, et al. Hand, foot and mouth disease caused by coxsackievirus A6, Thailand, 2012. Emerg. Infect. Dis. 2013;19:641–643. doi: 10.3201/eid1904.121666. PubMed DOI PMC

Wu Y, et al. The largest outbreak of hand; foot and mouth disease in Singapore in 2008: the role of enterovirus 71 and coxsackievirus A strains. Int. J. Infect. Dis. 2010;14:e1076–e1081. doi: 10.1016/j.ijid.2010.07.006. PubMed DOI

Feder HM, Bennett N, Modlin JF. Atypical hand, foot, and mouth disease: a vesiculobullous eruption caused by Coxsackie virus A6. Lancet Infect. Dis. 2014;14:83–A86. doi: 10.1016/S1473-3099(13)70264-0. PubMed DOI

Lott JP, et al. Atypical hand-foot-and-mouth disease associated with coxsackievirus A6 infection. J. Am. Acad. Dermatol. 2013;69:736–741. doi: 10.1016/j.jaad.2013.07.024. PubMed DOI PMC

Montes, M. et al. Hand, foot, and mouth disease outbreak and coxsackievirus A6, northern Spain, 2011. Emerg. Infect. Dis.19, 676–678 (2013). PubMed PMC

Sinclair, C. et al. Atypical hand, foot, and mouth disease associated with coxsackievirus A6 infection, Edinburgh, United Kingdom, January to February 2014. Euro Surveill.19, 20745 (2014). PubMed

Drago F, Ciccarese G, Broccolo F, Rebora A, Parodi A. Atypical hand, foot, and mouth disease in adults. J. Am. Acad. Dermatol. 2017;77:e51–e56. doi: 10.1016/j.jaad.2017.03.046. PubMed DOI

Yang X, et al. Clinical features and phylogenetic analysis of severe hand-foot-and-mouth disease caused by Coxsackievirus A6. Infect. Genet. Evol. 2020;77:104054. doi: 10.1016/j.meegid.2019.104054. PubMed DOI

Blomqvist S, et al. Co-circulation of coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland. J. Clin. Virol. 2010;48:49–54. doi: 10.1016/j.jcv.2010.02.002. PubMed DOI

Broccolo F, et al. Severe atypical hand-foot-and-mouth disease in adults due to coxsackievirus A6: Clinical presentation and phylogenesis of CV-A6 strains. J. Clin. Virol. 2019;110:1–6. doi: 10.1016/j.jcv.2018.11.003. PubMed DOI

Jiang P, Liu Y, Ma H-C, Paul AV, Wimmer E. Picornavirus morphogenesis. Microbiol. Mol. Biol. Rev. 2014;78:418–437. doi: 10.1128/MMBR.00012-14. PubMed DOI PMC

Hogle JM. Poliovirus cell entry: common structural themes in viral cell entry pathways. Annu. Rev. Microbiol. 2002;56:677–702. doi: 10.1146/annurev.micro.56.012302.160757. PubMed DOI PMC

Harutyunyan S, et al. Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A) tail at the 3′-end. PLoS Pathog. 2013;9:e1003270. doi: 10.1371/journal.ppat.1003270. PubMed DOI PMC

Buchta D, et al. Enterovirus particles expel capsid pentamers to enable genome release. Nat. Commun. 2019;10:1138. doi: 10.1038/s41467-019-09132-x. PubMed DOI PMC

Korant BD, Lonberg-Holm K, Noble J, Stasny JT. Naturally occurring and artificially produced components of three rhinoviruses. Virology. 1972;48:71–86. doi: 10.1016/0042-6822(72)90115-8. PubMed DOI

Fricks CE, Hogle JM. Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding. J. Virol. 1990;64:1934–1945. doi: 10.1128/jvi.64.5.1934-1945.1990. PubMed DOI PMC

Plevka P, Perera R, Cardosa J, Kuhn RJ, Rossmann MG. Crystal structure of human enterovirus 71. Science. 2012;336:1274. doi: 10.1126/science.1218713. PubMed DOI PMC

Wang X, et al. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat. Struct. Mol. Biol. 2012;19:424. doi: 10.1038/nsmb.2255. PubMed DOI PMC

Ren J, et al. Structures of coxsackievirus A16 capsids with native antigenicity: implications for particle expansion, receptor binding, and immunogenicity. J. Virol. 2015;89:10500–10511. doi: 10.1128/JVI.01102-15. PubMed DOI PMC

Xu L, et al. Atomic structures of coxsackievirus A6 and its complex with a neutralizing antibody. Nat. Commun. 2017;8:505. doi: 10.1038/s41467-017-00477-9. PubMed DOI PMC

Chen J, et al. A 3.0-angstrom resolution cryo-electron microscopy structure and antigenic sites of coxsackievirus A6-like particles. J. Virol. 2018;92:e01257–01217. PubMed PMC

Lee, H. et al. The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs. Sci. Adv.2, e1501929 (2016). PubMed PMC

Belnap DM, et al. Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus. J. Virol. 2000;74:1342–1354. doi: 10.1128/JVI.74.3.1342-1354.2000. PubMed DOI PMC

Oberste MS, Penaranda S, Maher K, Pallansch MA. Complete genome sequences of all members of the species Human enterovirus A. J. Gen. Virol. 2004;85:1597–1607. doi: 10.1099/vir.0.79789-0. PubMed DOI

Chapman, M. S. & Liljas, L. in Advances in Protein Chemistry (Academic Press, 2003). PubMed

Krupovic M, Koonin EV. Multiple origins of viral capsid proteins from cellular ancestors. Proc. Natl Acad. Sci. USA. 2017;114:E2401–E2410. doi: 10.1073/pnas.1621061114. PubMed DOI PMC

Wien MW, Curry S, Filman DJ, Hogle JM. Structural studies of poliovirus mutants that overcome receptor defects. Nat. Struct. Biol. 1997;4:666–674. doi: 10.1038/nsb0897-666. PubMed DOI

Smyth M, Pettitt T, Symonds A, Martin J. Identification of the pocket factors in a picornavirus. Arch. Virol. 2003;148:1225–1233. doi: 10.1007/s00705-002-0974-4. PubMed DOI

Lewis JK, Bothner B, Smith Thomas J, Siuzdak G. Antiviral agent blocks breathing of the common cold virus. Proc. Natl Acad. Sci. USA. 1998;95:6774–6778. doi: 10.1073/pnas.95.12.6774. PubMed DOI PMC

Oliveira MA, et al. The structure of human rhinovirus 16. Structure. 1993;1:51–68. doi: 10.1016/0969-2126(93)90008-5. PubMed DOI

Chow M, et al. Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature. 1987;327:482–486. doi: 10.1038/327482a0. PubMed DOI

Moscufo N, Simons J, Chow M. Myristoylation is important at multiple stages in poliovirus assembly. J. Virol. 1991;65:2372–2380. doi: 10.1128/jvi.65.5.2372-2380.1991. PubMed DOI PMC

Scouras AD, Daggett V. The dynameomics rotamer library: amino acid side chain conformations and dynamics from comprehensive molecular dynamics simulations in water. Protein Sci. 2011;20:341–352. doi: 10.1002/pro.565. PubMed DOI PMC

Zhu, R. et al. Discovery and structural characterization of a therapeutic antibody against coxsackievirus A10. Sci. Adv.4, eaat7459 (2018). PubMed PMC

Chen J, et al. Coxsackievirus A10 atomic structure facilitating the discovery of a broad-spectrum inhibitor against human enteroviruses. Cell Discov. 2019;5:4. doi: 10.1038/s41421-018-0073-7. PubMed DOI PMC

Foo DGW, et al. Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides. Virus Res. 2007;125:61–68. doi: 10.1016/j.virusres.2006.12.005. PubMed DOI

Gao F, et al. Enterovirus 71 viral capsid protein linear epitopes: identification and characterization. Virol. J. 2012;9:26. doi: 10.1186/1743-422X-9-26. PubMed DOI PMC

Borley, D. W. et al. Evaluation and use of in-silico structure-based epitope prediction with foot-and-mouth disease virus. PLoS ONE8, e61122 (2013). PubMed PMC

Wang L, et al. Bioinformatics-based prediction of conformational epitopes for Enterovirus A71 and Coxsackievirus A16. Sci. Rep. 2021;11:5701. doi: 10.1038/s41598-021-84891-6. PubMed DOI PMC

Hadfield AT, et al. The refined structure of human rhinovirus 16 at 2.15 A resolution: implications for the viral life cycle. Structure. 1997;5:427–441. doi: 10.1016/S0969-2126(97)00199-8. PubMed DOI

Chandler-Bostock R, et al. Assembly of infectious enteroviruses depends on multiple, conserved genomic RNA-coat protein contacts. PLoS Pathog. 2020;16:e1009146. doi: 10.1371/journal.ppat.1009146. PubMed DOI PMC

Wilson KA, Holland DJ, Wetmore SD. Topology of RNA-protein nucleobase-amino acid pi-pi interactions and comparison to analogous DNA-protein pi-pi contacts. RNA. 2016;22:696–708. doi: 10.1261/rna.054924.115. PubMed DOI PMC

Lentz KN, et al. Structure of poliovirus type 2 Lansing complexed with antiviral agent SCH48973: comparison of the structural and biological properties of the three poliovirus serotypes. Structure. 1997;5:961–978. doi: 10.1016/S0969-2126(97)00249-9. PubMed DOI

Jeong E, Kim H, Lee S-W, Han K. Discovering the interaction propensities of amino acids and nucleotides from protein-RNA complexes. Mol. Cells. 2003;16:161–167. PubMed

Schmidt NJ, Ho HH, Lennette EH. Propagation and isolation of group A coxsackieviruses in RD cells. J. Clin. Microbiol. 1975;2:183–185. doi: 10.1128/jcm.2.3.183-185.1975. PubMed DOI PMC

Rueckert, R. R. in Comparative Virology (eds Maramorosch, K. & Kurstak, E.) (Academic Press, 1971).

Flint, S. J., Enquist, L. W., Racaniello, V. R. & Skalka, A. M. Principles of Virology: Molecular Biology, Pathogenesis, and Control of Animal Viruses 2nd edn (ASM Press, 2004).

Harland J, Brown SM. HSV growth, preparation, and assay. Methods Mol. Med. 1998;10:1–8. PubMed

Watson DH, Russell WC, Wildy P. Electron microscopic particle counts on herpes virus using the phosphotungstate negative staining technique. Virology. 1963;19:250–260. doi: 10.1016/0042-6822(63)90062-X. PubMed DOI

Carpenter JE, Henderson EP, Grose C. Enumeration of an extremely high particle-to-PFU ratio for Varicella-zoster virus. J. Virol. 2009;83:6917–6921. doi: 10.1128/JVI.00081-09. PubMed DOI PMC

Klasse PJ. Molecular determinants of the ratio of inert to infectious virus particles. Prog. Mol. Biol. Transl. Sci. 2015;129:285–326. doi: 10.1016/bs.pmbts.2014.10.012. PubMed DOI PMC

Liu Y, et al. Structure and inhibition of EV-D68, a virus that causes respiratory illness in children. Science. 2015;347:71–74. doi: 10.1126/science.1261962. PubMed DOI PMC

Smith TJ, et al. The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating. Science. 1986;233:1286–1293. doi: 10.1126/science.3018924. PubMed DOI

Curry S, Chow M, Hogle JM. The poliovirus 135S particle is infectious. J. Virol. 1996;70:7125–7131. doi: 10.1128/jvi.70.10.7125-7131.1996. PubMed DOI PMC

Zhao Y, et al. Hand-foot-and-mouth disease virus receptor KREMEN1 binds the canyon of Coxsackie Virus A10. Nat. Commun. 2020;11:38. doi: 10.1038/s41467-019-13936-2. PubMed DOI PMC

Xu, L. et al. Cryo-EM structures reveal the molecular basis of receptor-initiated coxsackievirus uncoating. Cell Host Microbe29, 448–462.e5 (2021). PubMed PMC

Hrebik, D. et al. ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating. Proc. Natl Acad. Sci. USA118, e2024251118 (2021). PubMed PMC

R Core Team. R: a language and environment for statistical computing. (Vienna, Austria, 2018).

Kärber G. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn-Schmiedebergs Arch. f.ür. experimentelle Pathologie und Pharmakologie. 1931;162:480–483. doi: 10.1007/BF01863914. DOI

Zheng SQ, et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods. 2017;14:331–332. doi: 10.1038/nmeth.4193. PubMed DOI PMC

Zhang K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 2016;193:1–12. doi: 10.1016/j.jsb.2015.11.003. PubMed DOI PMC

Tang G, et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 2007;157:38–46. doi: 10.1016/j.jsb.2006.05.009. PubMed DOI

Wagner T, et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2019;2:218. doi: 10.1038/s42003-019-0437-z. PubMed DOI PMC

Zivanov J, et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife. 2018;7:e42166. doi: 10.7554/eLife.42166. PubMed DOI PMC

Vilas JL, et al. MonoRes: automatic and accurate estimation of local resolution for electron microscopy maps. Structure. 2018;26:337–344.e334. doi: 10.1016/j.str.2017.12.018. PubMed DOI

Ramírez-Aportela E, et al. Automatic local resolution-based sharpening of cryo-EM maps. Bioinformatics. 2020;36:765–772. PubMed PMC

de la Rosa-Trevín JM, et al. Xmipp 3.0: an improved software suite for image processing in electron microscopy. J. Struct. Biol. 2013;184:321–328. doi: 10.1016/j.jsb.2013.09.015. PubMed DOI

de la Rosa-Trevín JM, et al. Scipion: a software framework toward integration, reproducibility and validation in 3D electron microscopy. J. Struct. Biol. 2016;195:93–99. doi: 10.1016/j.jsb.2016.04.010. PubMed DOI

Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 1996;116:71–76. doi: 10.1006/jsbi.1996.0013. PubMed DOI

Winn MD, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D. 2011;67:235–242. doi: 10.1107/S0907444910045749. PubMed DOI PMC

Pettersen EF, et al. UCSF chimera - a visualization system for exploratory research and analysis. J. Comput. Chem. 2004;25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr. Sect. D. 2010;66:486–501. doi: 10.1107/S0907444910007493. PubMed DOI PMC

Liebschner D, et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. Sect. D. 2019;75:861–877. doi: 10.1107/S2059798319011471. PubMed DOI PMC

Murshudov GN, et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. Sect. D. 2011;67:355–367. doi: 10.1107/S0907444911001314. PubMed DOI PMC

Chen VB, et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D. 2010;66:12–21. doi: 10.1107/S0907444909042073. PubMed DOI PMC

Wiederstein M, Gruber M, Frank K, Melo F, Sippl MJ. Structure-based characterization of multiprotein complexes. Structure. 2014;22:1063–1070. doi: 10.1016/j.str.2014.05.005. PubMed DOI PMC

Wiederstein M, Sippl MJ. TopMatch-web: pairwise matching of large assemblies of protein and nucleic acid chains in 3D. Nucleic Acids Res. 2020;48:W31–W35. doi: 10.1093/nar/gkaa366. PubMed DOI PMC

Kabsch W. A solution for the best rotation to relate two sets of vectors. Acta Crystallogr. Sect. A. 1976;32:922–923. doi: 10.1107/S0567739476001873. DOI

Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 2007;372:774–797. doi: 10.1016/j.jmb.2007.05.022. PubMed DOI

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evolution. 2013;30:772–780. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Gouet P, Courcelle E, Stuart DI, Metoz F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics. 1999;15:305–308. doi: 10.1093/bioinformatics/15.4.305. PubMed DOI

Crameri, F. Scientific colour maps. Zenodo (2018).

Brewer, C. A. Colorbrewer colour maps. https://colorbrewer2.org/ (2020).

Pettersen EF, et al. UCSF chimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 2021;30:70–82. doi: 10.1002/pro.3943. PubMed DOI PMC

Schindelin J, et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Croll TI. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr D. Struct. Biol. 2018;74:519–530. doi: 10.1107/S2059798318002425. PubMed DOI PMC

Endosome rupture enables enteroviruses from the family Picornaviridae to infect cells