We have determined the structure of a human rhinovirus (HRV)-Fab complex by using cryoelectron microscopy and image reconstruction techniques. This is the first view of an intact human virus complexed with a monoclonal Fab (Fab17-IA) for which both atomic structures are known. The surface area on HRV type 14 (HRV14) in contact with Fab17-IA was approximately 500 A2 (5 nm2), which is much larger than the area that constitutes the NIm-IA epitope (on viral protein VP1) defined by natural escape mutants. From modeling studies and electrostatic potential calculations, charged residues outside the neutralizing immunogenic site IA (NIm-IA) were also predicted to be involved in antibody recognition. These predictions were confirmed by site-specific mutations and analysis of the Fab17-IA-HRV14 complex, along with knowledge of the crystallographic structures of HRV14 and Fab17-IA. The bound Fab17-IA reaches across a surface depression (the canyon) and meets a related Fab at the nearest icosahedral twofold axis. By adjusting the elbow angles of the bound Fab fragments from 162 degrees to 198 degrees, an intact antibody molecule can be easily modeled. This, along with aggregation and binding stoichiometry results, supports the earlier proposal that this antibody binds bivalently to the surface of HRV14 across icosahedral twofold axes. One prediction of this model, that the intact canyon-spanning immunoglobulin G molecule would block attachment of the virus to HeLa cells, was confirmed experimentally.

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.

Previous molecular and immunological studies have mapped four neutralization sites on human rhinovirus type 14 (B. Sherry, A. G. Mosser, R. J. Colonno, and R. R. Rueckert, J. Virol. 57:246-257, 1986). Eight monoclonal antibodies, one pair for each of the four target sites and all belonging to a single isotype, immunoglobulin G2a, were studied under conditions which resulted in 95% neutralization of infectious viral particles. All eight antibodies shifted the isoelectric point of virions from 6.7 to much more acidic forms, ranging from pl 1.8 to 3.2. In addition, antibodies targeted against three of the four neutralization sites caused significant aggregation of virions under the neutralization conditions employed. Aggregation could be reversed by digesting virus-antibody complexes with papain. Following papain digestion, the acidic pls of three of the neutralized virus preparations returned to neutral and infectivity was restored. Membrane-binding assays with virus neutralized with a nonaggregating antibody showed a dose-related inhibition of virus attachment to cellular receptors. Purified Fab fragments at a 13to 61-fold-higher concentration than intact antibodies caused a comparable isoelectric shift, neutralized virions in the absence of aggregation, and interfered with attachment of virions to host cell receptors in a membrane-binding assay. These findings suggest that neutralizing antibodies interfere with the attachment of rhinoviruses to cellular receptors and that bivalent attachment of antibody is not a prerequisite for neutralization.on July 6, 2020 by guest

Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.