Alison Burman

Integrin αvβ8 Functions as a Receptor for Foot-and-Mouth Disease Virus: Role of the β-Chain Cytodomain in Integrin-Mediated Infection

ABSTRACT Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three αv integrins, αvβ1, αvβ3, and αvβ6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human β8 cDNA and expression of αvβ8 at the cell surface. The involvement of αvβ8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the αvβ8 heterodimer or the αv chain. Similar results were obtained with a chimeric αvβ8 including the β6 cytodomain (αvβ8/6), showing that the β6 cytodomain can substitute efficiently for the corresponding region of β8. In contrast, virus binding to αvβ6 including the β8 cytodomain (αvβ6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the αvβ6 chimera was recognized poorly by antibodies specific for the ectodomain of αvβ6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the β6 cytodomain is important for maintaining αvβ6 in a conformation required for productive infection by FMDV.

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.

Foot-and-Mouth Disease Virus Forms a Highly Stable, EDTA-Resistant Complex with Its Principal Receptor, Integrin αvβ6: Implications for Infectiousness

ABSTRACT The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin αvβ6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD +1 and RGD +4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to αvβ6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin αvβ6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.

Foot-and-Mouth Disease Virus Induces Autophagosomes during Cell Entry via a Class III Phosphatidylinositol 3-Kinase-Independent Pathway

ABSTRACT Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, ∼50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity.

Highlights ► Efficient expression of FMDV empty capsids in insect cells after moderation of 3C protease action. ► Expression cassette productive in multiple insect cell lines. ► Empty capsids visualised by transmission electron microscopy. ► Empty capsids react with wide range of positive sera as well as authentic virus. ► Efficient empty capsid synthesis may allow development as a vaccine.

Guinea Pig-Adapted Foot-and-Mouth Disease Virus with Altered Receptor Recognition Can Productively Infect a Natural Host

ABSTRACT We report that adaptation to infect the guinea pig did not modify the capacity of foot-and-mouth disease virus (FMDV) to kill suckling mice and to cause an acute and transmissible disease in the pig, an important natural host for this pathogen. Adaptive amino acid replacements (I248→T in 2C, Q44→R in 3A, and L147→P in VP1), selected upon serial passages of a type C FMDV isolated from swine (biological clone C-S8c1) in the guinea pig, were maintained after virus multiplication in swine and suckling mice. However, the adaptive replacement L147→P, next to the integrin-binding RGD motif at the GH loop in VP1, abolished growth of the virus in different established cell lines and modified its antigenicity. In contrast, primary bovine thyroid cell cultures could be productively infected by viruses with replacement L147→P, and this infection was inhibited by antibodies to αvβ6 and by an FMDV-derived RGD-containing peptide, suggesting that integrin αvβ6 may be used as a receptor for these mutants in the animal (porcine, guinea pig, and suckling mice) host. Substitution T248→N in 2C was not detectable in C-S8c1 but was present in a low proportion of the guinea pig-adapted virus. This substitution became rapidly dominant in the viral population after the reintroduction of the guinea pig-adapted virus into pigs. These observations illustrate how the appearance of minority variant viruses in an unnatural host can result in the dominance of these viruses on reinfection of the original host species.

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

A role for endoplasmic reticulum exit sites in foot-and-mouth disease virus infection

Picornaviruses replicate their genomes in association with cellular membranes. While enteroviruses are believed to utilize membranes of the early secretory pathway, the origin of the membranes used by foot-and-mouth disease virus (FMDV) for replication are unknown. Secretory-vesicle traffic through the early secretory pathway is mediated by the sequential acquisition of two distinct membrane coat complexes, COPII and COPI, and requires the coordinated actions of Sar1, Arf1 and Rab proteins. Sar1 is essential for generating COPII vesicles at endoplasmic reticulum (ER) exit sites (ERESs), while Arf1 and Rab1 are required for subsequent vesicle transport by COPI vesicles. In the present study, we have provided evidence that FMDV requires pre-Golgi membranes of the early secretory pathway for infection. Small interfering RNA depletion of Sar1 or expression of a dominant-negative (DN) mutant of Sar1a inhibited FMDV infection. In contrast, a dominant-active mutant of Sar1a, which allowed COPII vesicle formation but inhibited the secretory pathway by stabilizing COPII coats, caused major disruption to the ER-Golgi intermediate compartment (ERGIC) but did not inhibit infection. Treatment of cells with brefeldin A, or expression of DN mutants of Arf1 and Rab1a, disrupted the Golgi and enhanced FMDV infection. These results show that reagents that block the early secretory pathway at ERESs have an inhibitory effect on FMDV infection, while reagents that block the early secretory pathway immediately after ER exit but before the ERGIC and Golgi make infection more favourable. Together, these observations argue for a role for Sar1 in FMDV infection and that initial virus replication takes place on membranes that are formed at ERESs.

Integrin sub-unit expression in cell cultures used for the diagnosis of foot-and-mouth disease☆

The ability to propagate foot-and-mouth disease virus (FMDV) plays an important role in laboratory diagnosis and the production of vaccines to control the spread of the disease. Many established cell lines suffer from poor sensitivity for isolating virus from field samples. One possible factor that limits sensitivity to FMDV is the lack of expression of surface integrins, the primary class of cell receptor used by FMDV to initiate infection. In this study we have sequenced cDNAs encoding these molecules for pigs and subsequently developed quantitative real-time reverse transcription (RT)-PCR assays to quantify underlying mRNA transcription of integrin molecules. These novel assays were used together with flow-cytometry to determine cell surface expression and of 4 different cell culture systems. These studies have identified a clear correlation of sensitivity to FMDV with expression of integrins αVβ6 and αVβ8. In contrast, cell surface expression of αVβ3 or mRNA for the β1, β3 or β5 subunits did not appear to contribute to sensitivity of cells to FMDV. These findings confirm the requirement for αV6 and αVβ8 as receptors for isolating FMDV from clinical samples and provide important tools and information for the rational design of recombinant cell lines containing these ligands for improved FMDV diagnosis and vaccine production.