Antibody-Mediated Neutralization of Human Rhinovirus 14 Explored by Means of Cryoelectron Microscopy and X-Ray Crystallography of Virus-Fab Complexes

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the β-B–β-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.Picornaviruses are among the largest of animal virus families and include the well-known poliovirus, rhinovirus, foot-and-mouth disease virus (FMDV), coxsackievirus, and hepatitis A virus. The rhinoviruses, of which there are more than 100 serotypes subdivided into two groups, are major causative agents of the common cold in humans (42). The viruses are nonenveloped and have an ∼300-Å-diameter protein shell that encapsidates a single-stranded, plus-sense RNA genome of about 7,200 bases. The human rhinovirus 14 (HRV14) capsid exhibits a pseudo-T=3 (P=3) icosahedral symmetry and consists of 60 copies each of four viral proteins, VP1, VP2, VP3, and VP4, with VP4 at the RNA-capsid interface (40). An ∼20-Å deep canyon lies roughly at the junction of VP1 (forming the north rim) with VP2 and VP3 (forming the south rim) and surrounds each of the 12 icosahedral fivefold vertices. The canyon regions of HRV14 and HRV16, both major receptor group rhinoviruses, were shown to contain the binding site of the cellular receptor, intercellular adhesion molecule 1 (ICAM-1) (8, 24a, 37). Four major neutralizing immunogenic (NIm) sites, NIm-IA, NIm-IB, NIm-II, and NIm-III, were identified by studies of neutralization escape mutants with monoclonal antibodies (MAbs) (46, 47) and then mapped to four protruding regions on the viral surface (40).Several mechanisms of antibody-mediated neutralization have been proposed. Perhaps the simplest is based on aggregation of virions (5, 53, 54), which generally occurs over a narrow range of antibody/virus ratios. This limited range has raised questions about the role of aggregation in vivo. Alternative suggestions are that antibodies may neutralize virions by inducing extensive conformational changes in the capsid (15, 29), abrogate virus attachment to the host cell (8, 14), or prevent uncoating (57). There is no universal acceptance of a single neutralization mechanism, and the various MAbs may neutralize with different combinations of these mechanisms.Neutralizing MAbs against HRV14 have been divided into three groups: strong, intermediate, and weak neutralizers (26, 34). All strongly neutralizing antibodies bind to the NIm-IA site, which was defined by natural escape mutations at residues D1091 and E1095 of VP1 on the loop between the β-B and β-C strands of the VP1 β-barrel (the letter designates the amino acid, the first digit identifies the viral protein, and the remaining three digits specify the sequence number). Because strongly neutralizing antibodies form stable, monomeric virus-antibody complexes with a maximum stoichiometry of 30 antibodies per virion, it was concluded that they bind bivalently to the virions (26, 34). Weakly neutralizing antibodies form unstable, monomeric complexes with HRV14 and bind with a stoichiometry of ∼60 antibodies per virion (26, 52). The remaining antibodies, all of which precipitate the virions, are classified as intermediate neutralizers (26, 34).The structures of two complexes, the strongly neutralizing antibody MAb17-IA and its Fab fragment, Fab17, bound to HRV14, were determined by means of cryo-transmission electron microscopy (cryo-TEM) and three-dimensional image reconstruction (51, 52) and interpreted on the basis of model-building studies that used the atomic structures of HRV14 (40) and Fab17 (28). These studies showed that no observable conformational changes were induced in the viral capsid upon Fab or MAb binding. Modeling and site-directed mutagenesis studies demonstrated that electrostatic interactions play a key role in the binding of Fab17 to HRV14 (52). In the complex, the loop of the NIm-IA site on HRV14 sits clamped in the cleft between the heavy- and light-chain hypervariable regions and forms complementary electrostatic interactions with Lys58^H (on the heavy chain) and Arg91^L (on the light chain) of Fab17. In addition, a cluster of lysines on HRV14 (K1236, K1097, and K1085) interact with two acidic residues, Asp45^H and Asp54^H, in the CDR2 (CDR stands for complementarity-determining region) of the Fab heavy chain (49). Earlier modeling studies also suggested that bidentate binding of MAb17-IA to HRV14 is facilitated by rotation of the Fab constant domains about the elbow axes towards the viral twofold axes (51). This suggested that the flexibility of the elbow region (the junction between the variable and constant domains) plays a role in the bivalent binding process, which in turn increases antibody avidity. Finally, the 4-Å-resolution crystal structure of the Fab17-HRV14 complex clearly showed that the virion does not undergo conformational changes upon Fab binding (49). This crystal structure determination also revealed that the earlier docking of the HRV14 and Fab17 atomic structures into the 22-Å cryo-TEM density map (50) yielded a pseudo-atomic model that was very close to the real structure of the complex.We have expanded our complementary X-ray crystallography and cryo-TEM microscopy studies to examine the structures of two more Fab-virus complexes, using Fab fragments from two other NIm-IA antibodies, MAb1-IA (MAb1) and MAb12-IA (MAb12), bound to HRV14. MAb1 and MAb12 are weak and strong neutralizing antibodies, respectively. Image reconstructions of these two complexes are interpreted on the basis of pseudo-atomic models, which substantiate the previous hypothesis that neutralizing efficacy and binding valency are interrelated (34). Electrostatic interactions at the epitope-paratope interface are highly conserved and apparently important for the antibody binding to the virion surface. Like Fab17, Fab1 and Fab12 penetrate the canyon. There are, however, differences between the orientations of the strongly and weakly neutralizing antibodies and in the contacts made with the receptor binding region of the canyon. Finally, data suggesting that antibody binding to HRV14 is alone sufficient for neutralization and that other possible mechanisms are not required are presented.