Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three ␣v integrins, ␣v␤1, ␣v␤3, and ␣v␤6, 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 ␤8 cDNA and expression of ␣v␤8 at the cell surface. The involvement of ␣v␤8 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 ␣v␤8 heterodimer or the ␣v chain. Similar results were obtained with a chimeric ␣v␤8 including the ␤6 cytodomain (␣v␤8/6), showing that the ␤6 cytodomain can substitute efficiently for the corresponding region of ␤8. In contrast, virus binding to ␣v␤6 including the ␤8 cytodomain (␣v␤6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the ␣v␤6 chimera was recognized poorly by antibodies specific for the ectodomain of ␣v␤6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the ␤6 cytodomain is important for maintaining ␣v␤6 in a conformation required for productive infection by FMDV.Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease, a severe vesicular disease of cloven-hoofed animals including domesticated ruminants and pigs. The virus exists as seven serotypes, which are members of the genus Aphthovirus of the family Picornaviridae (35). The virion consists of an 8.5-kb strand of RNA enclosed within an icosahedral capsid formed from 60 copies each of four proteins, VP1 to VP4 (1).Two classes of cell surface receptors that mediate FMDV infection have been identified (30). Theses are the integrins (7, 31, 33) and heparan sulfate (HS) proteoglycans (HSPGs) (29). The ability to use HSPGs as receptors appears to be restricted to strains of FMDV that have been multiply passaged through cultured cell lines (4,5,22,41,52,58), and presently there is no convincing evidence of a role for HS in cell entry by field viruses. Instead, field viruses are dependent on integrin receptors to initiate infection in vitro, and integrins are believed to be the receptors used in the infected animal. Recently, two independent studies have shown that certain strains of FMDV can infect cultured cells via an entry pathway that is independent of both integrins and cellular HS, implying the existence of a third, as yet unidentified receptor family (4, 65).Integrins are a family of integral membrane receptors with distinct ligand-binding specificities and tissue distributions. They contribute to a variety of cellular functions, including cell-cell and cell-matrix adhesion, and exist in alternative lowand high-affinity states, enab...

We have shown that foot-and-mouth disease virus (FMDV) infection mediated by the integrin ␣v␤6 takes place through clathrin-dependent endocytosis but not caveolae or other endocytic pathways that depend on lipid rafts. Inhibition of clathrin-dependent endocytosis by sucrose treatment or expression of a dominantnegative version of AP180 inhibited virus entry and infection. Similarly, inhibition of endosomal acidification inhibited an early step in infection. Blocking endosomal acidification did not interfere with surface expression of ␣v␤6, virus binding to the cells, uptake of the virus into endosomes, or cytoplasmic virus replication, suggesting that the low pH within endosomes is a prerequisite for delivery of viral RNA into the cytosol. Using immunofluorescence confocal microscopy, FMDV colocalized with ␣v␤6 at the cell surface but not with the B subunit of cholera toxin, a marker for lipid rafts. At 37°C, virus was rapidly taken up into the cells and colocalized with markers for early and recycling endosomes but not with a marker for lysosomes, suggesting that infection occurs from within the early or recycling endosomal compartments. This conclusion was supported by the observation that FMDV infection is not inhibited by nocodazole, a reagent that inhibits vesicular trafficking between early and late endosomes (and hence trafficking to lysosomes). The integrin ␣v␤6 was also seen to accumulate in early and recycling endosomes on virus entry, suggesting that the integrin serves not only as an attachment receptor but also to deliver the virus to the acidic endosomes. These findings are all consistent with FMDV infection proceeding via clathrin-dependent endocytosis.

SignificanceAn understanding of the mechanisms by which viruses evade host immunity is essential to the development of antiviral drugs and viral detection strategies. Ubiquitin and ubiquitin-like modifications are crucial in cellular innate immune and infection responses and are often suppressed by viral proteins. We here identify a previously unknown mechanism of viral evasion. A viral protease, Lbpro, removes ubiquitin and the ubiquitin-like protein ISG15 incompletely from proteins. While this strategy efficiently and irreversibly shuts down these modification systems, it enables repurposing of tools and technologies developed for ubiquitin research in virus detection. Specifically, we show that foot-and-mouth disease virus infection can be detected using an anti-GlyGly antibody developed for ubiquitin mass spectrometry research.

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 ␣v␤6. 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 ␣v␤6 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 ␣v␤6. 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 (FMDV) is the type species of the genus Aphthovirus within the family Picornaviridae and the etiological agent of foot-and-mouth disease, a severe vesicular condition affecting a large number of artiodactyls, including domesticated ruminants and pigs (1, 34). Presently, the virus is endemic in many parts of the world, including South America, Africa, and Asia (19). Foot-and-mouth disease is highly contagious and difficult to control as FMDV has a wide host range (see above) and a rapid replication cycle, small amounts of virus can initiate infection, and infected animals excrete high levels of virus. In addition, multiple modes of transmission have been recognized, including airborne spread, sometimes over long distances, including overseas (1,10,12,31).Field isolates of FMDV use integrins to initiate infection (14, 15, 29). The integrin family of cell adhesion receptors are a conserved series of ␣␤ heterodimers, which bind in a divalent cation-dependent manner to ligands through recognition of short motifs that usually include one of the acidic residues glutamate (E) or aspartate (D) (13). Examples of such motifs include arginine-glycine-aspartate (RGD) or leucine-aspartate-valine (LDV), and short peptides containing these motifs can interact similarly with integrins (13). Recognition of RGDcontaining proteins can proceed in a stepwise manner where the initial RGD binding is enhanced by a second stabilizing interaction involving so-called synergy sites on the ligand (2, 21, 23). The concept of a synergy site was first described for binding of ␣5␤1 to fibronectin (Fn). Thus, high-affinity binding of Fn to ␣5␤1 requires the RGD motif located on the 10th type III domain of Fn and a second synergy site in the 9th type III domain (23). Similarly, the large extracellular matrix protein ...

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, ϳ50% 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. Macroautophagy is a highly conserved degradation pathway that recycles cytoplasmic organelles and proteins in response to starvation (48). During macroautophagy (here referred to as autophagy), cup-shaped isolation membranes expand to form double-membrane autophagosomes that engulf cytosolic cargo and then fuse with late endosomes or lysosomes for degradation (49). Autophagosome formation is regulated by the mTORC1 (mammalian target of rapamycin complex 1) kinase, which is a negative regulator of autophagy. Autophagy can therefore be activated in the absence of starvation by mTORC1 inhibitors, such as rapamycin (46) or torin (43). mTORC1 regulates a multimeric complex (the ULK-1 complex) made up of ULK-1 (unc-51-like kinase), FIP200 (focal adhesion kinase family interacting protein of 200 kDa), Atg13, and Atg101 (4, 8-11, 13, 17, 26). Other key autophagy components, such as the phosphatidylinositol 3-kinase (PI3-kinase) complex (Atg6/beclin-Atg14-Vps15-Vps34) and the Atg12 (Atg12-Atg5-Atg16) and LC3 conjugation systems operate downstream of ULK-1 (13, 47). Increased PI3-kinase activity leads to recruitment of the Atg12-Atg5-Atg16 complex to isolation membranes and lipidation of LC3 to generate LC3II. Membrane association of LC3II facilitates expansion of the isolatio...

Foot-and-mouth disease virus (FMDV) can use a number of different integrins (␣v␤1, ␣v␤3, ␣v␤6, and ␣v␤8) as receptors to initiate infection. Infection mediated by ␣v␤6 is known to occur by clathrinmediated endocytosis and is dependent on the acidic pH within endosomes. On internalization, virus is detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in late endosomal compartments. Due to the extreme sensitivity of FMDV to acidic pH, it is thought that EE can provide a pH low enough for infection to occur; however, definitive proof that infection takes place from within these compartments is still lacking. Here we have investigated the intracellular transport steps required for FMDV infection of IBRS-2 cells, which express ␣v␤8 as their FMDV receptor. These experiments confirmed that FMDV infection mediated by ␣v␤8 is also dependent on clathrin-mediate endocytosis and an acidic pH within endosomes. Also, the effect on FMDV infection of dominant-negative (DN) mutants of cellular rab proteins that regulate endosomal traffic was examined. Expression of DN rab5 reduced the number of FMDV-infected cells by 80%, while expression of DN rab4 or DN rab7 had virtually no effect on infection. Expression of DN rab11 inhibited infection by FMDV, albeit to a small extent (ϳ35%). These results demonstrate that FMDV infection takes place predominantly from within EE and does not require virus trafficking to the late endosomal compartments. However, our results suggest that infection may not be exclusive to EE and that a small amount of infection could occur from within PNRE. Foot-and-mouth disease virus (FMDV) is a member of theAphthovirus genus of the family Picornaviridae and the etiological agent responsible for FMD, an economically important and severe vesicular condition of cloven-hoofed animals, including cattle, pigs, sheep, and goats (2). The mature virus particle consists of a positive-sense single-stranded RNA genome (vRNA) enclosed within a nonenveloped icosahedral capsid formed from 60 copies each of four virus-encoded proteins, VP1 to VP4 (1).The initial stage of FMDV infection is virus binding to cell surface integrins via a highly conserved RGD motif located on the GH loop of VP1. A number of different species of RGDbinding integrins (␣v␤1, ␣v␤3, ␣v␤6, and ␣v␤8) have been reported to serve as receptors for FMDV (5,(23)(24)(25)(26). Using pharmacological and dominant-negative (DN) inhibitors of specific endocytic pathways in combination with immunofluorescence confocal microscopy, the cell entry pathway used by FMDV has been determined for ␣v␤6-expressing cells (6, 36). These studies established that infection occurs by clathrinmediated endocytosis and is dependent on the acidic pH within endosomes, which serves as the trigger for capsid disassembly and translocation of the vRNA across the endosomal membrane into the cytosol. Internalized virus was detected rapidly in early endosomes (EE) and subsequently in perinuclear recycling endosomes (PNRE), but not in l...

Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived from the O/UKG/34/2001 or A/Turkey 2/2006 field viruses, were constructed using the backbone from the O1K B64 cDNA, and viable viruses (O1K/O-UKG and O1K/A-Tur, respectively) were successfully rescued in each case. These viruses grew well in primary bovine thyroid cells but grew less efficiently in BHK cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs of foot-and-mouth disease were observed, which then spread to in-contact animals. Thus, the surface-exposed capsid proteins of the O1K B64 strain are responsible for its attenuation in cattle. Consequently, there is no evidence for any adaptation, acquired during cell culture, outside the capsid coding region within the O1K B64 strain that inhibits replication in cattle. These chimeric infectious cDNA plasmids provide a basis for the analysis of FMDV pathogenicity and characterization of receptor utilization in vivo.

Field isolates of foot-and-mouth disease virus (FMDVF oot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most widespread, epizootic transboundary animal diseases, affecting many species of wildlife and livestock, such as cattle, sheep, goats, and pigs. The significant economic losses that result from FMD are due to the high morbidity of infected animals and stringent trade restrictions imposed on affected countries (1). Vaccination plays a major role in controlling FMD, either to lessen the effects of an outbreak in FMDfree countries or for control and eradication in regions where it is endemic. The etiological agent of FMD, foot-and-mouth disease virus (FMDV), exists as seven distinct serotypes (O, A, C, Asia-1, and the Southern African Territories [SAT] serotypes SAT-1, SAT-2, and SAT-3). Within each serotype, a large number of antigenic variants exist (2). Intraserotype diversity is driven by a high mutation rate during replication that is caused by an error-prone viral RNA-dependent RNA polymerase (3) and thus complicates efforts to control disease by vaccination due to incomplete protection between some antigenic variants (4). Hence, the most effective vaccines closely match the outbreak virus, which can necessitate the development of new vaccine strains. The current vaccines are inactivated virus preparations grown in large-scale cell culture. Therefore, the production of a new vaccine is critically dependent upon adaptation of viruses from the field for growth in cell culture, which can prove problematical for some viruses.Foot-and-mouth disease virus is the type species of the Aphthovirus genus of the Picornaviridae, a family of nonenveloped, single-stranded positive-sense RNA viruses. The viral capsid is formed by 60 copies each of four structural proteins (VP1 to VP4) arranged in icosahedral symmetry. The outer capsid surfaces are formed by VP1, which surrounds the five-fold symmetry axis, and VP2 and VP3, which alternate around the three-fold axis (5). VP4 is myristoylated and located inside the capsid and is thought to play an essential role in the final stage of assembly and in endosomal membrane penetration by the viral RNA (6, 7). In vivo, FMDV has a strong tropism for epithelial cells, which is in part due to the epithelial cell-restricted expression of integrin ␣v␤6, which is the principal receptor used by field viruses to initiate infection (8-12). Integrin binding is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) motif located at the apex of a structurally disordered loop (the GH loop of VP1). The integrin specificity of FMDV has been the subject of several studies, and three other RGD-dependent integrins (␣v␤1, ␣v␤3, and ␣v␤8) have also been reported to be receptors for field strains of the virus (13-15); however, the role of these integrins in pathogenesis is unclear, and we have found that ␣v␤3 is a poor receptor for FMDV in vitro (16). Furthermore, despite recognizing their ligands via the RGD motif, two other RGD-dependent integrins ...