Antibodies to the Vitronectin Receptor (Integrin a V b 3 ) Inhibit Binding and Infection of Foot-and-Mouth Disease Virus to Cultured Cells (original) (raw)
Journal of …, 1995
The amino acid sequence Arg-Gly-Asp (RGD) is highly conserved on the VP1 proteins of different serotypes and subtypes of foot-and-mouth disease virus (FMDV) and is essential for cell attachment. This sequence is also found in certain extracellular matrix proteins that bind to a family of cell surface receptors called integrins. Within the Picornaviridae family, human enterovirus coxsackievirus A9 also has an RGD motif on its VP1 capsid protein and has recently been shown to utilize the vitronectin receptor integrin ␣ V  3 as a receptor on monkey kidney cells. Competition binding experiments between type A 12 FMDV and coxsackievirus A9 using BHK-21 and LLC-MK2 cells revealed shared receptor specificity between these two viruses. Polyclonal antiserum to the vitronectin receptor and a monoclonal antibody to the ␣ V subunit inhibited both FMDV binding and plaque formation, while a monoclonal antibody to the  3 subunit inhibited virus binding. In contrast, antibodies to the fibronectin receptor (␣ 5  1) or to the integrin (␣ V  5) had no effect on either binding or plaque formation. These data demonstrate that the ␣ V  3 vitronectin receptor can function as a receptor for FMDV.
Journal of Virology, 2008
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 ␣v6. 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 ␣v6 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 ␣v6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.
Journal of Comparative Pathology, 2009
Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals principally affecting cattle, pigs and sheep. FMD virus (FMDV) uses the a V b 1 , a V b 3 , a V b 6 , and a V b 8 integrins as receptors in vitro via a highly conserved arginineeglycineeaspartic acid amino acid sequence motif located within the bG-bH loop of VP1. Immunofluorescence and confocal microscopy were used to study the expression of two major FMDV receptors, a V b 3 and a V b 6 , within epithelial tissues from FMDV-infected and uninfected cattle in order to understand the role of these receptors in tissue tropism. Integrin a V b 6 was expressed by epithelial cells in tissues that are important sites for FMDV replication (i.e. tongue and coronary band). Integrin a V b 3 was detected in epithelium of all tissues examined except tongue. In addition, a V b 3 expression was associated with blood vessels in all tissues examined. In infected tissues, a V b 6 integrin was distributed on the surface of those epithelial cells also expressing FMDV antigen. Although integrin a V b 3 has been shown to be a receptor for FMDV, no expression of a V b 3 was associated with FMDV-positive keratinocytes in the tongue. In contrast, podal epithelial cells containing FMDV antigen also expressed a V b 3 integrin. Thus, at the cellular level the expression of these two integrins correlates with susceptibility to infection and may contribute substantially to viral tropism in FMD pathogenesis.
2020
Infection of foot-and-mouth disease virus (FMDV) is initiated by binding to cellular receptor molecules of the integrin family. Integrin αvβ3 has been identified as one of the RGD-binding integrins. The aim of this study is the determination of αvβ3 integrin receptor expression variations during subsequent passages of BHK-21 suspension cell cultures on the A-type FMDV quality. Anchorage dependent BHK-21 cells were adapted to grow in submerged system suspension cell culture, and with subsequently cultivated for 40 serial passages in the bioreactor. During this period, the expression of αvβ3 integrin receptor was determined by the Western blotting technique in the intervals of subsequent cell culture passages before FMDV inoculation. Modifications of the plaque morphology during serial BHK-21 passages infected with FMDV were detected by plaque assay. Fluctuations and abnormal expression of integrin in BHK-21 suspension cells were found to be associated with the number of passages in suspension culture. These changes took place on the surface properties of cells, and significant reduction was observed in the αvβ3 integrin receptor expression, where it also negatively affected the FMDV plaque characters. This study will provide a better quality FMD vaccine production with higher immunity and more extended efficiency properties. Besides that, it is also a pioneering study in the diagnosis of other viruses which use integrin receptors and their respective vaccines.
Journal of General Virology, 2005
Field strains of Foot-and-mouth disease virus (FMDV) use a number of av-integrins as receptors to initiate infection on cultured cells, and integrins are believed to be the receptors used to target epithelial cells in animals. In this study, immunofluorescence confocal microscopy and real-time RT-PCR were used to investigate expression of two of the integrin receptors of FMDV, avb6 and avb3, within various epithelia targeted by this virus in cattle. These studies show that avb6 is expressed constitutively on the surfaces of epithelial cells at sites where infectious lesions occur during a natural infection, but not at sites where lesions are not normally formed. Expression of avb6 protein at these sites showed a good correlation with the relative abundance of b6 mRNA. In contrast, avb3 protein was only detected at low levels on the vasculature and not on the epithelial cells of any of the tissues investigated. Together, these data suggest that in cattle, avb6, rather than avb3, serves as the major receptor that determines the tropism of FMDV for the epithelia normally targeted by this virus.
Journal of Virology, 1997
The integrin ␣v3 has been shown to act as the receptor for internalization of foot-and-mouth disease virus (FMDV) (A12), with attachment being through a highly conserved RGD motif located on the G-H loop of viral capsid protein VP1. In addition, however, we have recently shown that efficient infection of culture-grown cells by FMDV (O1BFS) requires binding to cell surface heparan sulfate. In this study, we have used a solid-phase receptor binding assay to characterize the binding by FMDV to purified ␣v3 in the absence of heparan sulfate and other cell surface components. In this assay, FMDV (O1BFS) successfully replicated authentic ligand binding by cellular ␣v3 in terms of its high affinity, dependence on divalent cations, and activation by manganese ions. Virus binding to this preparation of ␣v3 was exquisitely sensitive to competition by short RGDcontaining peptides (50% inhibition at <10 ؊8 M peptide), and this inhibition was highly sequence specific, with the equivalent RGE peptide being at least 10 4 fold less effective as a competitor. Representative viruses of the other six serotypes of FMDV bound to ␣v3 in a similar RGD-specific manner, although significant differences in sensitivity to RGD peptides suggest that the affinity of the different FMDV serotypes for ␣v3 is influenced, in part, by the variable amino acid residues in the VP1 G-H loop on either side of the RGD.
Cellular Receptors for Foot and Mouth Disease Virus
Intervirology, 2009
Foot-and-mouth disease virus (FMDV), the prototype member of the Aphthovirus genus, is a single-stranded, positivesense RNA genome virus, which affects many domestic livestock cloven-hoofed animals, causing substantial lost of milk in dairy cattle, reduction in the growth rate of meat animals, among others. It has been shown that the virus can enter to the cells using different pathways; the main one binding integrins via the clathrin-mediated endocytosis pathway, trafficking throughout the acidified endocytic vesicles, where its capsid rapidly dissociates, resulting in the release of the RNA genome, and the second one using heparan sulfate in which FMDV enters to the cells using the caveola-mediated endocytosis pathway and that caveolae can associate and traffic with endosomes. Different integrins had been involved as FMDV receptors (␣ v  1, ␣ v  3, ␣ 5  1, ␣ v  6, ␣ v  8); this review will try to resume the basic information about FMDV receptors from the last years to the present and will resume the most important in vitro and in vivo studies to elucidate the role of this receptor on the infection.
Analysis of Foot-and-Mouth Disease Virus Internalization Events in Cultured Cells
Journal of Virology, 2005
It has been demonstrated that foot-and-mouth disease virus (FMDV) can utilize at least four members of the αV subgroup of the integrin family of receptors in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the βG-βH loop of VP1. While there have been extensive studies of virus-receptor interactions at the cell surface, our understanding of the events during viral entry into the infected cell is still not clear. We have utilized confocal microscopy to analyze the entry of two FMDV serotypes (types A and O) after interaction with integrin receptors at the cell surface. In cell cultures expressing both the αVβ3 and αVβ6 integrins, virus adsorbed to the cells at 4°C appears to colocalize almost exclusively with the αVβ6 integrin. Upon shifting the infected cells to 37°C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular ...
Journal of Virology, 2000
Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-1433Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin ␣ v  3 . In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.
The Journal of general virology, 2014
In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 bE-bF loop, VP1 C-terminus and VP3 b-B knob) surrounding the fivefold symmetry axis (VP1 bF-bG loop) and at other discrete sites on the capsid (VP3 bG-bH loop, VP1 C-terminus, VP2 bC strand and VP1 bG-bH loop). A lysine insertion in the VP1 bF-bG loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 bG-bH loop ("3, "2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 bB-bC loop) suggest their possible role in modulating the efficiency and specificity of interaction of the 'RGD' motif with a v -integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.
Journal of virology, 1996
Foot-and-mouth disease virus (FMDV) enters cells by attaching to cellular receptor molecules of the integrin family, one of which has been identified as the RGD-binding integrin alpha(v)beta3. Here we report that, in addition to an integrin binding site, type O strains of FMDV share with natural ligands of alpha(v)beta3 (i.e., vitronectin and fibronectin) a specific affinity for heparin and that binding to the cellular form of this sulfated glycan, heparan sulfate, is required for efficient infection of cells in culture. Binding of the virus to paraformaldehyde-fixed cells was powerfully inhibited by agents such as heparin, that compete with heparan sulfate or by agents that compete for heparan sulfate (platelet factor 4) or that inactivate it (heparinase). Neither chondroitin sulfate, a structurally related component of the extracellular matrix, nor dextran sulfate appreciably inhibited binding. The functional importance of heparan sulfate binding was demonstrated by the facts that...
Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus
Journal of General Virology, 2015
Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1-VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A2). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A2 vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult.
Journal of Virology, 2003
In this work we analyze the antigenic properties and the stability in cell culture of virus mutants recovered upon challenge of peptide-vaccinated cattle with foot-and-mouth disease virus (FMDV) C3 Arg85. Previously, we showed that a significant proportion of 29 lesions analyzed (41%) contained viruses with single amino acid replacements (R141G, L144P, or L147P) within a major antigenic site located at the G-H loop of VP1, known to participate also in interactions with integrin receptors. Here we document that no replacements at this site were found in viruses from 12 lesions developed in six control animals upon challenge with FMDV C3 Arg85. Sera from unprotected, vaccinated animals exhibited poor neutralization titers against mutants recovered from them. Sequence analyses of the viruses recovered upon 10 serial passages in BHK-21 and FBK-2 cells in the presence of preimmune (nonneutralizing) sera revealed that mutants reverted to the parental sequence, suggesting an effect of the ...
The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex
The EMBO Journal, 1999
Heparan sulfate has an important role in cell entry by foot-and-mouth disease virus (FMDV). We find that subtype O 1 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 sulfatebinding 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.
Journal of Virology, 2003
After 13 passages on BHK cells to produce a vaccine, a Cathay topotype isolate of FMDV serotype O from China (O/CHA/90) extended its cell culture host range and bound to heparin-Sepharose, although it did not require cell surface HS as a receptor molecule. To understand these phenomena, we constructed chimeric viruses by using a type A 12 infectious cDNA and the capsid protein-coding regions of O/CHA/90 and its cell culture-adapted derivative (vac-O/CHA/90). Using a set of viruses derived from these chimeras by exchanging portions of the capsid-coding regions, we discovered that a group of amino acid residues that surround the fivefold axis of the icosahedral virion determine host range in cell culture and influence pathogenicity in pigs. These residues included aromatic amino acids at positions 108 and 174 and positively charged residues at positions 83 and 172 in protein 1D. To test if these residues participated in non-integrin-dependent cell binding, the integrin-binding RGD sequence in protein 1D was changed to KGE in two different chimeras. Evaluation of these KGE viruses indicated that growth in cell culture was not dependent on HS. One of these viruses was tested in pigs, where it produced a mild disease and maintained its KGE sequence. These results are discussed in terms of receptor utilization and pathogenesis of this important pathogen.
Journal of Virology, 2013
Field isolates of foot-and-mouth disease virus (FMDV) have a restricted cell tropism which is limited by the need for certain RGD-dependent integrin receptors. In contrast, cell culture-adapted viruses use heparan sulfate (HS) or other unidentified molecules as receptors to initiate infection. Here, we report several novel findings resulting from cell culture adaptation of FMDV. In cell culture, a virus with the capsid of the A/Turkey/2/2006 field isolate gained the ability to infect CHO and HS-deficient CHO cells as a result of a single glutamine (Q)-to-lysine (K) substitution at VP1-110 (VP1-Q 110 K ). Using site-directed mutagenesis, the introduction of lysine at this same site also resulted in an acquired ability to infect CHO cells by type O and Asia-1 FMDV. However, this ability appeared to require a second positively charged residue at VP1-109. CHO cells express two RGD-binding integrins (␣51 and ␣v5) that, although not used by FMDV, have the potential to be used as receptors; however, viruses with the VP1-Q 110 K substitution did not use these integrins. In contrast, the VP1-Q 110 K substitution appeared to result in enhanced interactions with ␣v6, which allowed a virus with KGE in place of the normal RGD integrin-binding motif to use ␣v6 as a receptor. Thus, our results confirmed the existence of nonintegrin, non-HS receptors for FMDV on CHO cells and revealed a novel, non-RGD-dependent use of ␣v6 as a receptor. The introduction of lysine at VP1-110 may allow for cell culture adaptation of FMDV by design, which may prove useful for vaccine manufacture when cell culture adaptation proves intractable.
Journal of virology, 2003
Adaptation of field isolates of foot-and-mouth disease virus (FMDV) to grow in cells in culture can result in changes in viral properties that include acquisition of the ability to bind to cell surface heparan sulfate (HS). After 13 passages on BHK cells to produce a vaccine, a Cathay topotype isolate of FMDV serotype O from China (O/CHA/90) extended its cell culture host range and bound to heparin-Sepharose, although it did not require cell surface HS as a receptor molecule. To understand these phenomena, we constructed chimeric viruses by using a type A 12 infectious cDNA and the capsid protein-coding regions of O/CHA/90 and its cell culture-adapted derivative (vac-O/CHA/90). Using a set of viruses derived from these chimeras by exchanging portions of the capsid-coding regions, we discovered that a group of amino acid residues that surround the fivefold axis of the icosahedral virion determine host range in cell culture and influence pathogenicity in pigs. These residues included aromatic amino acids at positions 108 and 174 and positively charged residues at positions 83 and 172 in protein 1D. To test if these residues participated in non-integrin-dependent cell binding, the integrin-binding RGD sequence in protein 1D was changed to KGE in two different chimeras. Evaluation of these KGE viruses indicated that growth in cell culture was not dependent on HS. One of these viruses was tested in pigs, where it produced a mild disease and maintained its KGE sequence. These results are discussed in terms of receptor utilization and pathogenesis of this important pathogen.
Journal of Clinical Microbiology, 2013
Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the α V β 6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the α V and β 6 bovine integrin subunits. This stable cell line (LFBK-α V β 6 ) showed β 6 expression and enhanced susceptibility to FMDV infection for ≥100 cell passages. LFBK-α V β 6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types t...