Canine parvovirus (CPV) is a highly contagious pathogen that triggers severe disease in dogs and wildlife. of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the disease capsid MK-0859 that control receptor binding. The antibody footprint is definitely markedly different from the previous one recognized by using a 12 ? structure. This work emphasizes the need for any high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time. Intro The characteristics of a successful sponsor antibody response and the producing neutralization mechanisms are poorly recognized despite the key role played by antibodies in protecting animals against disease infection. Disease neutralization may be accomplished through one or more mechanisms, which can differ widely, depending on the specific antibody. Neutralization mechanisms that have been defined include cross-linking or aggregation of capsids (1, 2), prevention of uncoating (3, 4), interference with receptor binding by sterically obstructing the virus-receptor connection (5,C7), and induction of a capsid conformational switch (8, 9). A better understanding of these and additional specific antibody-virus relationships will be useful for improving vaccines that elicit protecting antibodies. Canine parvovirus (CPV) is definitely a highly contagious disease that causes severe enteritis in dogs and crazy carnivores, and antibodies are a important component of protecting immunity to this disease (10, 11). The single-stranded DNA genome is definitely packaged into a nonenveloped icosahedral capsid 26 nm in diameter. The disease is put together with a major capsid protein, VP2, and a few copies of a minor structural proteins, VP1, which consists of a distinctive N-terminal peptide that’s packaged inside the capsid interior. All the capsid subunits consist of an eight-stranded antiparallel -barrel theme commonly within many disease structural protein. Loops linking the strands constitute a lot of the CPV surface area. Distinctive spikes encircling each icosahedral 3-collapse axis mediate sponsor receptor reputation and antigenicity (12,C15). The CPV capsid binds to transferrin receptor type 1 on its sponsor cells, counting on extremely particular relationships with residues from the 3-fold spike (12, 13, 16, 17). A -panel of eight antibodies to CPV once was characterized by utilizing a structural evaluation to check binding and neutralization research. A number of the Fabs, including Fab E, had MK-0859 been proven to neutralize the disease at nonsaturating concentrations of <20 Fab substances per capsid, whereas others had been poorly neutralizing actually at high concentrations of Fab substances per binding site (18). The cryo-electron microscopy (cryo-EM) reconstructions previously referred to provided virus-Fab complicated maps which range from 9 to MK-0859 18 ? in quality (19). Web-based antibody versions (WAM) had been utilized to interpret these maps also to determine the antibody-binding areas or footprints for the capsid external (20). Nevertheless, the 12 ? quality cryo-EM map from the CPV capsid-Fab E complicated did not display the interaction at length or reveal a MK-0859 particular mechanism of neutralization (19). Improvements in microscopy, image detection, and software have made it possible to obtain cryo-EM structures of atomic and near-atomic resolution, allowing the building of atomic models (21,C23). Here we redefine the CPV capsid-Fab E complex by solving the structure at near-atomic resolution. Virus building was initiated by first fitting the crystal structure, and the Fab was built into density. The residues in the interaction interface were unambiguously identified even though steric collisions limit the binding of Fab to one Fab molecule per icosahedral 2-fold axis. A local resolution map revealed that Fab binding induced significant changes in Rabbit Polyclonal to MAP3KL4. the nearby 3-fold spikes, which became poorly resolved likely because of flexibility that does not exist in this region in wild-type virus structures. Thus, the high-resolution map reported here has provided additional information that reveals a likely mechanism of MAb and Fab neutralization. MATERIALS AND METHODS Cryo-EM data collection. Capsids were purified by sucrose gradient centrifugation as previously described (12). The IgGs were purified from hybridoma supernatants by protein G chromatography, and the Fab was isolated after digestion with pepsin as described previously (18). Purified Fab E was incubated with CPV capsids at a ratio of four Fab molecules to MK-0859 each of the 60.