Elizabeth E. Fry

Fitness Cost of Escape Mutations in p24 Gag in Association with Control of Human Immunodeficiency Virus Type 1

ABSTRACT Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.

The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex.

Heparan sulfate has an important role in cell entry by foot‐and‐mouth disease virus (FMDV). We find that subtype O1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3. Two pre‐formed sulfate‐binding sites control receptor specificity. Residue 56 of VP3, an arginine in this virus, is critical to this recognition, forming a key component of both sites. This residue is a histidine in field isolates of the virus, switching to an arginine in adaptation to tissue culture, forming the high affinity heparan sulfate‐binding site. We postulate that this site is a conserved feature of FMDVs, such that in the infected animal there is a biological advantage to low affinity, or more selective, interactions with glycosaminoglycan receptors.

A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71

Enterovirus 71 (EV71) is a major agent of hand, foot and mouth disease in children that can cause severe central nervous system disease and death. No vaccine or antiviral therapy is available. High-resolution structural analysis of the mature virus and natural empty particles shows that the mature virus is structurally similar to other enteroviruses. In contrast, the empty particles are markedly expanded and resemble elusive enterovirus-uncoating intermediates not previously characterized in atomic detail. Hydrophobic pockets in the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. These structures provide a model for enterovirus uncoating in which the VP1 GH loop acts as an adaptor-sensor for cellular receptor attachment, converting heterologous inputs to a generic uncoating mechanism, highlighting new opportunities for therapeutic intervention.

The structure and antigenicity of a type C foot-and-mouth disease virus.

BACKGROUND Picornaviruses are responsible for a wide range of mammalian diseases and, in common with other RNA viruses, show considerable antigenic variation. Foot-and-mouth disease viruses (FMDVs) constitute one genus of the picornavirus family and are classified into seven serotypes, each of which shows considerable intratypic variation. This antigenic variation leads to continuing difficulties in controlling the disease. To date the structure of only one serotype, O, has been reported. RESULTS The three-dimensional structure of a serotype C (isolate C-S8c1) FMDV, has been determined crystallographically at 3.5 A resolution. The main chain conformation of the virion is very similar to that of type O1 virus. The immunodominant G-H loop of VP1, the presumed site of cell attachment, is disordered in both types of virus indicating a functional role for flexibility of this region. There are significant changes in the structure of other antigenic loops and in some internal regions involved in protomer-protomer contacts, including the entire amino-terminal portion of VP2, described here for the first time for a picornavirus. Antigenic sites have been identified by genetic and peptide mapping methods, and located on the capsid. The data reveal a major new discontinuous antigenic site (site D) which is located near to the three-fold axis and involves residues of VP1, VP2 and VP3 which lie adjacent to each other on the capsid. CONCLUSION In FMDV type C, amino acid substitutions seen in mutants that are resistant to neutralization by monoclonal antibodies (MAbs) map to predominantly surface-oriented residues with solvent-accessible side-chains not involved in interactions with other amino acids, whereas residues which are accessible but not substituted are found to be more frequently involved in protein-protein interactions. This provides a molecular interpretation for the repeated isolation of the same amino acid substitutions in MAb-resistant variants, an observation frequently made with RNA viruses. This first comparison of two FMDV serotypes shows how subtle changes at antigenic sites are sufficient to cause large changes in antigenic specificity between serotypes.

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single β-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

Picornavirus uncoating intermediate captured in atomic detail.

It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release.

In situ macromolecular crystallography using microbeams

A sample environment for mounting crystallization trays has been developed on the microfocus beamline I24 at Diamond Light Source. The technical developments and several case studies are described.

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.

Specificity of the VP1 GH Loop of Foot-and-Mouth Disease Virus for αv Integrins

ABSTRACT Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by αvβ6 or αvβ8 but not for inhibition of virus binding to αvβ3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for αvβ6. In contrast, although RGDM-containing peptides were effective for αvβ8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses αvβ8 less efficiently than αvβ6 as a receptor for infection. Finally, evidence is presented that shows αvβ3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to αvβ6 with a higher affinity than to αvβ3 and αvβ8.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen.

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.