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Neutralizing Antibodies Can Initiate Genome Release from Human Enterovirus 71

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Neutralizing Antibodies Can Initiate Genome Release from Human Enterovirus 71 Neutralizing antibodies can initiate genome release from human enterovirus 71 Pavel Plevkaa,1, Pei-Yin Limb, Rushika Pereraa,2, Jane Cardosab,c, Ampa Suksatua, Richard J. Kuhna, and Michael G. Rossmanna,3 aDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907; bSentinext Therapeutics, 10050 Penang, Malaysia; and cMAB Explorations, 10050 Penang, Malaysia Edited by Wah Chiu, Baylor College of Medicine, Houston, TX, and approved January 6, 2014 (received for review November 7, 2013) Antibodies were prepared by immunizing mice with empty, imma- Here we present an analysis of the interactions of the mono- ture particles of human enterovirus 71 (EV71), a picornavirus that clonal antibodies E18 and E19 with EV71. By using cryo-EM, we causes severe neurological disease in young children. The capsid show that binding of E18 to EV71 causes the virus to change its structure of these empty particles is different from that of the conformation to the A state and to eject much of its genome. mature virus and is similar to “A” particles encountered when This was further verified by fluorescence activation when SYBR picornaviruses recognize a potential host cell before genome release. Green dyes interact with RNA. In contrast, although mAb E19 The monoclonal antibody E18, generated by this immunization, does neutralize the virus, it has a quite different footprint on the induced a conformational change when incubated at temperatures virus surface and does not cause ejection of the genome. between 4 °C and 37 °C with mature virus, transforming infectious virions into A particles. The resultant loss of genome that was Results and Discussion observed by cryo-EM and a fluorescent SYBR Green dye assay The E18 and E19 antibodies were prepared by immunizing mice inactivated the virus, establishing the mechanism by which the with empty, immature EV71 particles containing VP0 (18). Both virus is inactivated and demonstrating that the E18 antibody has E18 and E19 could neutralize the virus as intact antibodies or as potential as an anti-EV71 therapy. The antibody-mediated virus Fab fragments (Fig. 1). Both these mAbs can recognize confor- neutralization by the induction of genome release has not been mational epitopes on the surface of heat-inactivated EV71 par- previously demonstrated. Furthermore, the present results indicate ticles by indirect ELISA. However, these antibodies could not that antibodies with genome-release activity could also be produced recognize linear epitopes by using immunoblot analysis (Fig. 2). for other picornaviruses by immunization with immature particles. The Fab fragments of these mAbs were incubated with EV71 for cryo-EM studies of the mAb–virion complexes. Visual inspection nterovirus 71 (EV71) is a picornavirus that causes hand, foot, of the cryo-EM micrographs showed that as many as 20% of the Eand mouth disease (1). In infants and small children, the EV71 particles that had been incubated with E18 had lost much infection may proceed to encephalitis that can be fatal or result or all of their RNA genome (Fig. 3 A, D, E, and F). In contrast in permanent brain damage. EV71 virions are nonenveloped with a diameter of approximately 300 Å. The capsid has icosa- Significance hedral, pseudo-T=3 symmetry with four viral proteins VP1, VP2, VP3, and VP4 in each icosahedral asymmetric unit (2, 3). Sub- Enterovirus 71 (EV71) causes yearly outbreaks of hand, foot, units VP1, VP2, and VP3 have a jelly-roll fold common to many and mouth disease in Southeast Asian countries including viruses. VP4 is a small protein attached to the inner face of the China and Malaysia. Some of the infected children develop capsid. EV71 infections produce fully infectious RNA-filled encephalitis that can be fatal or result in permanent brain particles and empty immature particles that lack genome and damage. There are no anti-EV71 therapeutic agents available. contain capsid protein VP0, the precursor of VP4 and VP2 (3). Here it is shown that an antibody that had been generated by These empty particles have approximately 5% larger diameter using an immature EV71 virus as an antigen induced the re- than the mature virions. Furthermore, the protomer formed by lease of genome from EV71 virions, rendering the virus non- VP0, VP1, and VP3 is rotated by 5.4° relative to the protomer infectious. The induction of genome release is a mechanism formed by VP1, VP2, VP3, and VP4 in the mature particle with by which antibodies can neutralize viruses. Furthermore, the respect to the icosahedral symmetry axes. The empty particles approach presented in the paper could be used to prepare are presumably precursors of the mature infectious virions (3). antibodies with similar properties against related viruses that Rhino and entero picornaviruses have a depression, called the include significant human pathogens. “canyon,” on the virion surface encircling the icosahedral five- fold axes (4). The canyon is frequently the site of binding of Author contributions: P.P. and M.G.R. designed research; P.P., P.-Y.L., R.P., J.C., and A.S. picornavirus receptors (5–8), although some receptor molecules performed research; P.P., P.-Y.L., R.P., J.C., R.J.K., and M.G.R. wrote the paper. bind to other sites on picornavirus capsids (9, 10). Experimental The authors declare no conflict of interest. evidence indicates that binding of a receptor into the canyon This article is a PNAS Direct Submission. results in the expulsion of the “pocket factor” from the hydro- Data deposition: Cryo-EM reconstructions were deposited with the EM Data Bank, www. – emdatabank.org [accession numbers EMD-2397 (E18 full), EMD-2434 (E18 empty), and phobic cavity within VP1 (11 14). Ejection of the pocket factor EMD-2436 (E19 Fab–EV71)]. The atomic coordinates have been deposited in the Protein leads to destabilization of virions. Such activated “A” particles Data Bank, www.pdb.org [PDB ID codes 4C0U (E18 full), 4C0Y (E18 empty), and 4C10 are characterized by expansion of the capsid, release of VP4, and (E19 Fab–EV71). externalization of the VP1 N-termini (6). The organization of the 1Present address: Central European Institute of Technology, Masaryk University, 625 00 major capsid proteins in the A particle and in the immature Brno, Czech Republic. 2 empty particles are similar (3). Transition of the virion to the A Present address: Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, state is a prerequisite for the release of the genome (15). Heating CO 80523. of picornavirus particles to nonphysiological temperatures of 50 °C 3To whom correspondence should be addressed. E-mail: [email protected]. to 60 °C can also induce transformation of virions to the A state in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vitro (6, 16, 17). 1073/pnas.1320624111/-/DCSupplemental. 2134–2139 | PNAS | February 11, 2014 | vol. 111 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1320624111 Downloaded by guest on September 30, 2021 Although EV71 can be completely inhibited by E18, the electron micrographs show that not all particles had released their genomes (Fig. 3F). However, the virus particles would be inactivated even if only a small part of the genome were released from the virion and degraded by RNAses. Such particles, even though noninfectious, would be evaluated as genome containing in our analysis. Indeed, the electron density corresponding to the genome was lower in the EV71–E18 complex (minimum, −2.2, maximum, 1.1; average, −0.17) than in the EV71–E19 complex (minimum, −1.6; maximum, 2.0; average, −0.15), even though only Fig. 1. Neutralization of EV71 by monoclonal antibodies E18 and E19. full-looking particles were used to calculate the (full) EV71–E18 Whole IgG and Fab fragments of the monoclonal antibodies E18 (A) and E19 reconstruction. (B) were used to inhibit EV71 at different concentrations (x axis) by using a plaque reduction neutralization test. The red symbols represent whole Alternatively, the neutralization of EV71 by E18 might be antibody and the blue symbols represent Fab fragments. Inhibition of virus achieved not only by inducing genome release but also by other was represented as the percentage of plaques relative to plaques in the means such as preventing receptor binding. Indeed, the E18 control wells. We demonstrated neutralization of EV71 by Fab fragments as footprint on the virion surface includes Lys-149 of VP2 that has well as by whole IgG, with whole IgG being more efficient than Fab fragments. been implicated to have a role in attachment of EV71 to the P-selectin glycoprotein ligand-1 receptor (Fig. 6 and Fig. S2)(19). TheE18bindingsitesontheEV71capsidsarelocated to the action of E18 Fab, only approximately 1% of the EV71 between VP4–VP2–VP3–VP1 protomers (Fig. 6A and Table 2). particles incubated with E19 Fab had lost their genome (Fig. 3 However, the protomer in both A particles (after receptor B C and ). Thus, E18 but not E19 induced genome release binding) and empty, immature (before VP0 cleavage) particles is from virions. rotated by 5.4° relative to its position in the mature capsid with Separate reconstructions were made of the empty immature respect to the icosahedral axes (3, 16). Because the E18 antibody EV71–E18 complexes and of the full EV71–E18 complexes. The was generated by immunization with empty, immature particles, resolutions of these reconstructions were 10 Å and 20 Å, re- it is likely that, when E18 binds to mature EV71 particles, there spectively, as judged by the resolution at which the Fourier shell will occur an “induced fit” that requires local rearrangements of correlation coefficient decreased to less than 0.5 (Fig. S1). The the capsid to an immature-like capsid conformation.
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