Cryo-Electron Microscopy Study of Insect Cell-Expressed Enterovirus 71 and Coxsackievirus A16 Virus-Like Particles Provides a Structural Basis for Vaccine Development

Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in young children. Although several vaccines are currently in clinical trials, no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71-infected cells: an expanded empty capsid, sometimes called a procapsid, and the infectious virus. Specifically, an immunodominant epitope of EV71 that maps to the virus canyon is structurally different in the procapsid and virus. This structure-function study shows that the procapsid can sequester antibodies, thus enhancing EV71 infection in vitro. The results presented here suggest that, due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics. IMPORTANCEIn a picornavirus infection, both an infectious and a noninfectious empty capsid, sometimes referred to as a procapsid, are produced. It was novel to discover that the procapsid form of EV71 was expanded and antigenically distinct from the infectious virus. Previously, it had been supposed that this empty capsid was an off-pathway dead end or at best served for storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much noninfectious capsid. Here, we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus, the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics.

Section: Discussionmentioning

confidence: 99%

The Enterovirus 71 Procapsid Binds Neutralizing Antibodies and Rescues Virus Infection In Vitro

Shingler

Cifuente

Ashley

et al. 2015

“…Recently, Gong et al reported the cryo-EM structure of EV71 VLP produced in insect cells at 5.2-Å resolution (EMBL-EBI identifier EMD-2607) (19). The crystal structure of our EV71 VLP produced in yeast was fitted into the reported cryo-EM density map ( ϭ 1), which showed that the crystal structure agreed well with the cryo-EM structural features, except for a few protruding loops (cc ϭ 0.81).…”

Section: Ev71-vlp-c4 a S V Pmentioning

confidence: 99%

“…We used UCSF Chimera (18) to visualize the cryo-EM density map of EV71 VLP (EMD-2607) (19), to segment the corresponding density maps, and to compare the structural similarities between electron density and crystal structure of EV71 VLP. After docking the crystal structure of EV71 VLP (PDB ID 4YVS) into the EV71 VLP density map ( ϭ 1.00), the correlation coefficient (cc) values from comparisons of the cryo-EM density map and crystal structure were calculated using "Fit in Map" in UCSF Chimera (18).…”

Section: Figmentioning

confidence: 99%

Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease

Lyu

et al. 2015

Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth disease (HFMD). Previously, we demonstrated that a virus-like particle (VLP) for EV71 produced from Saccharomyces cerevisiae is a potential vaccine candidate against EV71 infection, and an EV71/CVA16 chimeric VLP can elicit protective immune responses against both virus infections. Here, we presented the crystal structures of both VLPs, showing that both the linear and conformational neutralization epitopes identified in EV71 are mostly preserved on both VLPs. The replacement of only 4 residues in the VP1 GH loop converted strongly negatively charged surface patches formed by portions of the SP70 epitope in EV71 VLP into a relatively neutral surface in the chimeric VLP, which likely accounted for the additional neutralization capability of the chimeric VLP against CVA16 infection. Such local variations in the amino acid sequences and the surface charge potential are also present in different types of polioviruses. In comparison to EV71 VLP, the chimeric VLP exhibits structural changes at the local site of amino acid replacement and the surface loops of all capsid proteins. This is consistent with the observation that the VP1 GH loop located near the pseudo-3-fold junction is involved in extensive interactions with other capsid regions. Furthermore, portions of VP0 and VP1 in EV71 VLP are at least transiently exposed, revealing the structural flexibility of the VLP. Together, our structural analysis provided insights into the structural basis of enterovirus neutralization and novel vaccine design against HFMD and other enterovirus-associated diseases. IMPORTANCEOur previous studies demonstrated that the enterovirus 71 (EV71) virus-like particle (VLP) produced from yeast is a vaccine candidate against EV71 infection and that a chimeric EV71/coxsackievirus A16 (CVA16) VLP with the replacement of 4 amino acids in the VP1 GH loop can confer protection against both EV71 and CVA16 infections. This study reported the crystal structures of both the EV71 VLP and the chimeric EV71/CVA16 VLP and revealed that the major neutralization epitopes of EV71 are mostly preserved in both VLPs. In addition, the mutated VP1 GH loop in the chimeric VLP is well exposed on the particle surface and exhibits a surface charge potential different from that contributed by the original VP1 GH loop in EV71 VLP. Together, this study provided insights into the structural basis of enterovirus neutralization and evidence that the yeast-produced VLPs can be developed into novel vaccines against hand-foot-and-mouth disease (HFMD) and other enterovirus-associated diseases. Hand-foot-and-mouth disease (HFMD) is a worldwide infectious disease in infants and young children that may lead to death. In recent years, numerous outbreaks of HFMD have occurred in the Asia-Pacific regions, causing significant morbidity and mortality (1). The number of reported fatal cases of HFMD in mainland China has reached over 3,000 since 2008 (www .c...

“…Recently, Ren et al reported crystal structures of the unexpanded CVA16 mature virion, the procapsid, and a recombinant virus-like particle (VLP) (12). In addition, the structure of the insect cell-produced CVA16 VLP has been determined at 5.5 Å by cryo-electron microscopy (cryo-EM) single-particle analysis (13). These studies reveal that the capsid of CVA16 virions is similar to those of other enteroviruses, which is an arrangement of 60 copies of protomers each consisting of 4 subunits known as VP1, VP2, VP3, and VP4.…”

mentioning

confidence: 99%

Beta-Propiolactone Inactivation of Coxsackievirus A16 Induces Structural Alteration and Surface Modification of Viral Capsids

Chen

et al. 2017

Beta-propiolactone (BPL) is an inactivating agent that is widely used in the vaccine industry. However, its effects on vaccine protein antigens and its mechanisms of action remain poorly understood. Here we present cryo-electron microscopy (cryo-EM) structures of BPL-treated coxsackievirus A16 (CVA16) mature virions and procapsids at resolutions of 3.9 Å and 6.5 Å, respectively. Notably, both particles were found to adopt an expanded conformation resembling the 135S-like uncoating intermediate, with characteristic features including an opened 2-fold channel, the externalization of the N terminus of VP1 capsid protein, and the absence of pocket factor. However, major neutralizing epitopes are very well preserved on these particles. Further biochemical analyses revealed that BPL treatment impairs the abilities of CVA16 particles to bind to the attachment receptor heparan sulfate and to a conformation-dependent monoclonal antibody in a BPL dose-dependent manner, indicating that BPL is able to modify surface-exposed amino acid residues. Taken together, our results demonstrate that BPL treatment may induce alteration of the overall structure and surface properties of a nonenveloped viral capsid, thus revealing a novel mode of action of BPL.IMPORTANCE Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. It is recognized that BPL inactivates viral infectivity through modification of viral nucleic acids. However, its effect on viral proteins remains largely unknown. Here, we present high-resolution cryo-EM structures of BPLtreated coxsackievirus A16 (CVA16) mature virions and procapsids, which reveals an expanded overall conformation and characteristic features that are typical for the 135S-like uncoating intermediate. We further show that the BPL concentration affects the binding of inactivated CVA16 particles to their receptor/antibody. Thus, BPL treatment can alter the overall structure and surface properties of viral capsids, which may lead to antigenic and immunogenic variations. Our findings provide important information for future development of BPL-inactivated vaccines.KEYWORDS Beta-propiolactone, coxsackievirus A16, cryo-EM structure, structural alteration, surface modification E nterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents of hand, foot, and mouth disease (HFMD), which has been prevalent in young children in the Asia-Pacific region (1). According to a recent survey, EV71 and CVA16 were associated with 50.4% and 38.3% of the 266 laboratory-confirmed HFMD