John Newman

Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop

Data from cryo‐electron microscopy and X‐ray crystallography have been combined to study the interactions of foot‐and‐mouth disease virus serotype C (FMDV‐C) with a strongly neutralizing monoclonal antibody (mAb) SD6. The mAb SD6 binds to the long flexible GH‐loop of viral protein 1 (VP1) which also binds to an integrin receptor. The structure of the virus–Fab complex was determined to 30 Å resolution using cryo‐electron microscopy and image analysis. The known structure of FMDV‐C, and of the SD6 Fab co‐crystallized with a synthetic peptide corresponding to the GH‐loop of VP1, were fitted to the cryo‐electron microscope density map. The SD6 Fab is seen to project almost radially from the viral surface in an orientation which is only compatible with monovalent binding of the mAb. Even taking into account the mAb hinge and elbow flexibility, it is not possible to model bivalent binding without severely distorting the Fabs. The bound GH‐loop is essentially in what has previously been termed the ‘up’ position in the best fit Fab orientation. The SD6 Fab interacts almost exclusively with the GH‐loop of VP1, making very few other contacts with the viral capsid. The position and orientation of the SD6 Fab bound to FMDV‐C is in accord with previous immunogenic data.

Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity

BACKGROUND Foot-and-mouth disease virus (FMDV) is an extremely infectious and antigenically diverse picornavirus of cloven-hoofed animals. Strains of the A22 subtype have been reported to change antigenically when adapted to different growth conditions. To investigate the structural basis of this phenomenon we have determined the structures of two variants of an A22 virus. RESULTS The structures of monolayer- and suspension-cell-adapted A22 FMDV have been determined by X-ray crystallography. Picornaviruses comprise four capsid proteins, VP1-4. The major antigenic loop of the capsid protein VP1 is flexible in both variants of the A22 subtype but its overall disposition is distinct from that observed in other FMDV serotypes (O and C). A detailed structural comparison between A22 FMDV and a type O virus suggests that different conformations in a portion of the major antigenic loop of VP1 (the GH loop, which is also central to receptor attachment) result in distinct folds of the adjacent VP3 GH loop. Also, a single mutation (Glu82-->Gly) on the surface of VP2 in the suspension-cell-adapted virus appears to perturb the structure of the VP1 GH loop. CONCLUSION The GH loop of VP1 is flexible in three serotypes of FMDV, suggesting that flexibility is important in both antigenic variability and structural communication with other regions of the virus capsid. Our results illustrate two instances of the propagation of structural perturbations across the virion surface: the change in the VP3 GH loop caused by the VP1 GH loop and the Glu82-->Gly change in VP2 which we believe perturbs the GH loop of VP1. In the latter case, the amplification of the sequence changes leads to differences, between the monolayer- and suspension-cell-adapted viruses, in host-cell interactions and antigenicity.

Structural comparison of two strains of foot-and-mouth disease virus subtype O1 and a laboratory antigenic variant, G67.

BACKGROUND Foot-and-mouth disease viruses (FMDVs) are members of the picornavirus family and cause an economically important disease of cloven-hoofed animals. To understand the structural basis of antigenic variation in FMDV, we have determined the structures of two viruses closely related to strain O1BFS whose structure is known. RESULTS The two new structure are, like O1BFS, both serotype O viruses. The first, O1 Kaüfbeuren (O1K), is a field isolate dating from an outbreak of FMD in Europe in the 1960s. The second, called G67, is a quadruple mutant of O1K, generated in the laboratory, that bears point mutations conferring resistance to neutralizing by monoclonal antibodies, specific for each of the four major antigenic sites defined previously. The availability of the three related virus structures permits a detailed analysis of the way amino acid substitutions influence antigenicity. Structural changes are seen to be limited, in general, to the substituted side chain. For example, the GH loop of VP1, a highly antigenic and mobile protuberance which becomes ordered only under reducing conditions, was essentially indistinguishable in the three viruses despite the accumulation of up to four changes within its 15-residue sequence. At one of the other antigenic sites, however, changes between the two field strains did perturb both side-chain and main-chain structures in the vicinity. CONCLUSIONS The conservation of conformation of the GH loop of VP1 adds to the evidence implicating an integrin as the cellular receptor for FMDV, since this loop contains a conserved RGD (Arg-Gly-Asp) sequence structurally similar to the same tripeptide in some other integrin-binding proteins. Structural changes required for the virus to escape neutralization by monoclonal antibodies are generally small. The more extensive type of structural change exhibited by the field isolates probably reflects differing selective pressures operating in vivo and in vitro.

Flexibility in the P2 domain of the HIV-1 Gag polyprotein.

The HIV‐1 Gag polyprotein contains a segment called p2, located between the capsid (CA) and nucleocapsid (NC) domains, that is essential for ordered virus assembly and infectivity. We subcloned, overexpressed, and purified a 156‐residue polypeptide that contains the C‐terminal capsid subdomain (CACTD) through the NC domain of Gag (CACTD‐p2‐NC, Gag residues 276–431) for NMR relaxation and sedimentation equilibrium (SE) studies. The CACTD and NC domains are folded as expected, but residues of the p2 segment, and the adjoining thirteen C‐terminal residues of CACTD and thirteen N‐terminal residues of NC, are flexible. Backbone NMR chemical shifts of these 40 residues deviate slightly from random coil values and indicate a small propensity toward an α‐helical conformation. The presence of a transient coil‐to‐helix equilibrium may explain the unusual and necessarily slow proteolysis rate of the CA‐p2 junction. CACTD‐p2‐NC forms dimers and self‐associates with an equilibrium constant (Kd = 1.78 ± 0.5 μM) similar to that observed for the intact capsid protein (Kd = 2.94 ± 0.8 μM), suggesting that Gag self‐association is not significantly influence by the P2 domain.

Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease viruses from serotypes O, A and C have been crystallized. The particular strains studied include O1K, A10(61), A22 Iraq 24/64, A24 Cruzeiro and C-S8c1. In addition, crystals have been grown of G67, a monoclonal antibody neutralization escape mutant derived from O1K, and of virus R100, recovered after the establishment of a persistent infection in baby hamster kidney cells with C-S8c1. Empty particles, capsids which lack the RNA genome, have also been crystallized for subtypes A22 Iraq 24/64 and A10(61). In almost all cases, crystals suitable for high resolution structure determination were obtained from (NH4)2SO4 or mixtures of polyethylene glycol and NH4Cl.

Qualitative thinking support systems (QTSS)

ABSTRACT The creative process of qualitative thinking is an integral part of decision making. There are information systems that support creative thinking and aspects of qualitative reasoning. These systems, however, are not designed to directly bolster qualitative thinking in the comprehensive, dynamic, and integrated manner required for effective decision making. A new framework, called a qualitative thinking support system (QTSS), can provide the desired support. This article demonstrates how QTSS can improve decision making. It overviews the decision making process, identifies gaps in information systems support for the qualitative aspects of this process, describes the QTSS concept and its role in closing the support gaps, and measures the impact of the QTSS on the process and outcomes of decision making. The paper also examines the implications of the analyses for information systems research and practice.