For every, 3 l of virus-Fab complex was pipetted onto a Quantifoil grid (Quantifoil Micro Tools GmbH, Jena, Germany), blotted to eliminate excess test, and plunge-frozen right into a liquid ethane-propane blend (25) using an Mk III Vitrobot (FEI, Hillsboro, OR). to lethal pulmonary edema and center failure (2), with mortality becoming saturated in youthful kids beneath the age group of 6 (2 specifically, 3). As seasonal outbreaks of HFMD are repeating across the global globe, advancement of a vaccine and antiviral therapies for EV71 is becoming an urgent concern. A member of the family, EV71 has a nonenveloped, icosahedral capsid comprised of 60 copies of each of four viral structural proteins (VP1 to VP4) (4). Recent studies have solved the constructions for three strains of EV71 (MY104 [5], Fuyang [6], and 1095 [7]), demonstrating that EV71 has the general features of picornavirus capsids, including the 5-fold mesa and the depression round the mesa called the canyon (5C8). Conserved residues VP1-242K and VP1-244K form positively charged patches within the 5-collapse mesa (6), and this symmetry-related clustering of positive costs has been suggested like a common mechanism for heparan sulfate binding in enteroviruses (9). Several cellular receptors for EV71 have been recognized: scavenger receptor B2 (SCARB2), P-selectin glycoprotein ligand-1 (PSGL-1), and heparan sulfate (HS) (10C12). SCARB2, which is definitely expressed on a broad variety of cell types, likely binds to the computer virus canyon and induces the transition of the virion that is required for uncoating (13C15). PSGL-1, which is definitely indicated specifically on lymphocytes, binds only specific EV71 strains and helps viral replication in lymphocytes inside a PSGL-1-dependent manner (11). Relating to recent studies, PSGL-1 and HS bind the positively charged patches within the 5-collapse mesa of EV71 and provide initial attachment within the cell (12, 16, 17). We recently found that the PSGL-1 binding phenotype of EV71 strains is definitely regulated by a single residue, VP1-145, that maps to the center of the 5-fold mesa (16). Linear epitopes of EV71 have been characterized in several Mitomycin C studies using synthetic peptides Mitomycin C or inactivated whole computer virus (18C21). The epitopes, which induce cross-neutralization activity, are located in the rim of the canyon on VP1 and VP2, where SCARB2 binds (18C20). Strain-specific neutralizing antibodies that identify conformation-dependent epitopes Mitomycin C have been generated and classified (22); however, the epitopes have not been mapped. Structural studies can determine and map the conformational epitopes and elucidate antibody neutralization mechanisms which support effective vaccine development. Here, we have characterized the EV71 monoclonal antibody (MAb) MA28-7 as having strain-specific neutralization activity. We used cryo-electron microscopy (cryo-EM) to solve the structure of EV71 complexed with the Fab fragment from MA28-7. The Fab fragment binds across the 5-fold symmetry axes such that steric hindrance limits occupation to no more than one Fab fragment per 5-fold vertex. The footprint of the Fab includes VP1-145 and residues that map to the positively charged patches (VP1-98, VP1-242, and VP1-244) round the 5-fold axis, overlapping Rabbit Polyclonal to EGR2 with the binding sites of PSGL-1 and HS. Mitomycin C Neutralization and sequence positioning analysis indicate that VP1-145 is definitely a determinant for the strain-specific antigenicity of EV71. MATERIALS AND METHODS Computer virus production. EV71 was propagated and purified as explained previously (8). Briefly, EV71 strain 1095 (23, 24) was propagated in HeLa cells for 24 h. The press and cells were collected and processed by freezing and thawing three times. Cell debris was.