A 3.0-Angstrom Resolution Cryo-Electron Microscopy Structure and Antigenic Sites of Coxsackievirus A6-Like Particles

Abstract

Coxsackievirus A6 (CVA6) has recently emerged as one of the predominant causative agents of hand, foot, and mouth disease (HFMD). The structure of the CVA6 mature viral particle has not been solved thus far. Our previous work shows that recombinant virus-like particles (VLPs) of CVA6 represent a promising CVA6 vaccine candidate. Here, we report the first cryo-electron microscopy (cryo-EM) structure of the CVA6 VLP at 3.0-Å resolution. The CVA6 VLP exhibits the characteristic features of enteroviruses but presents an open channel at the 2-fold axis and an empty, collapsed VP1 pocket, which is broadly similar to the structures of the enterovirus 71 (EV71) VLP and coxsackievirus A16 (CVA16) 135S expanded particle, indicating that the CVA6 VLP is in an expanded conformation. Structural comparisons reveal that two common salt bridges within protomers are maintained in the CVA6 VLP and other viruses of the genus, implying that these salt bridges may play a critical role in enteroviral protomer assembly. However, there are apparent structural differences among the CVA6 VLP, EV71 VLP, and CVA16 135S particle in the surface-exposed loops and C termini of subunit proteins, which are often antigenic sites for enteroviruses. By immunological assays, we identified two CVA6-specific linear B-cell epitopes (designated P42 and P59) located at the GH loop and the C-terminal region of VP1, respectively, in agreement with the structure-based prediction of antigenic sites. Our findings elucidate the structural basis and important antigenic sites of the CVA6 VLP as a strong vaccine candidate and also provide insight into enteroviral protomer assembly. Coxsackievirus A6 (CVA6) is becoming one of the major pathogens causing hand, foot, and mouth disease (HFMD), leading to significant morbidity and mortality in children and adults. However, no vaccine is currently available to prevent CVA6 infection. Our previous work shows that recombinant virus-like particles (VLPs) of CVA6 are a promising CVA6 vaccine candidate. Here, we present a 3.0-Å structure of the CVA6 VLP determined by cryo-electron microscopy. The overall architecture of the CVA6 VLP is similar to those of the expanded structures of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), but careful structural comparisons reveal significant differences in the surface-exposed loops and C termini of each capsid protein of these particles. In addition, we identified two CVA6-specific linear B-cell epitopes and mapped them to the GH loop and the C-terminal region of VP1, respectively. Collectively, our findings provide a structural basis and important antigenic information for CVA6 VLP vaccine development.

From Monovalent to Multivalent Vaccines, the Exploration for Potential Preventive Strategies Against Hand, Foot, and Mouth Disease (HFMD)

“…CV-A16 also has linear epitopes in the canyon floor of VP1 and the G-H loop of VP3 (Xu et al 2015;Fang and Liu 2018). Linear neutralizing antigenic sites were also reported in the E-F loop of CV-A10 VP2, in the G-H loop as well as the C-terminus of CV-A6 VP1 (Chen et al 2018;Dai et al 2019) Fig. 1 The structure of enterovirus 71 genome and virion organization.…”

“…CV-A16 also has linear epitopes in the canyon floor of VP1 and the G-H loop of VP3 (Xu et al 2015 ; Fang and Liu 2018 ). Linear neutralizing antigenic sites were also reported in the E–F loop of CV-A10 VP2, in the G-H loop as well as the C-terminus of CV-A6 VP1 (Chen et al 2018 ; Dai et al 2019 ).The identification of conserved neutralizing linear epitopes provides important targets for the development of multivalent vaccines (Xu et al 2015 ). For example, MAB979 is an antibody raised by EV-A71 immunization, and it recognized residues 136–150 of VP2 on extensive synthetic peptide screening.…”

“…Lee et al 2013;Kiener et al 2014;Jiang et al 2015;Arthur Huang et al 2017;Jia et al 2017). Studies on CV-A6 indicated that there are also conformational epitopes located in the B-C, E-F, H-I loops of VP1(Chen et al 2018;Fang and Liu 2018).…”

Outlier Profiles of Atomic Structures Derived from X-ray Crystallography and from Cryo-Electron Microscopy

“…Cryo-electron microscopy (cryo-EM) is an essential method to determine three-dimensional atomic structures of proteins and some RNA and DNA molecules [1][2][3][4][5][6][7]. Unlike X-ray crystallography, cryo-EM technique is not limited by crystallization of proteins, and hence it suits a broader range of molecules [8].…”

A Histogram-based Outlier Profile for Atomic Structures Derived from Cryo-Electron Microscopy

“…Cryo-electron microscopy (cryo-EM) is an essential method to determine three-dimensional atomic structures of proteins and some RNA and DNA molecules [1][2][3][4][5][6]. Cryo-EM technique is not limited by the crystallization step that is required in X-ray crystallography technique, and hence it suits broader types of molecules [7].…”