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Coxsackie B viruses (CVB) cause a wide spectrum of diseases, ranging from mild respiratory syndromes and hand, foot, and mouth disease to life-threatening conditions, such as pancreatitis, myocarditis, and encephalitis. Previously, we and others found that the soluble virus receptor trap sCAR-Fc strongly attenuates CVB3 infection in mice. In this study, we investigated whether treatment with sCAR-Fc results in development of resistance by CVB3. Two CVB3 strains (CVB3-H3 and CVB3 Nancy) were passaged in HeLa cells in the presence of sCAR-Fc. The CVB3-H3 strain did not develop resistance, whereas two populations of CVB3 Nancy mutants emerged, one with complete (CVB3 M ) and one with partial (CVB3 K ) resistance. DNA sequence alignment of the resistant virus variant CVB3 M with CVB3 Nancy revealed an amino acid exchange from Asn(N) to Ser(S) at position 139 of the CVB3 capsid protein VP2 (N2139S), an amino acid predicted to be involved in the virus's interaction with its cognate receptor CAR. Insertion of the N2139S mutation into CVB3-H3 by site-directed mutagenesis promoted resistance of the engineered CVB3-H3 N2139S to sCAR-Fc. Interestingly, development of resistance by CVB3-H3 N2139S and the exemplarily investigated CVB3 M -clone 2 (CVB3 M2 ) against soluble CAR did not compromise the use of cellular CAR for viral infection. Infection of HeLa cells showed that sCAR-Fc resistance, however, negatively affected both virus stability and viral replication compared to that of the parental strains. These data demonstrate that during sCAR-Fc exposure, CVB3 can develop resistance against sCAR-Fc by single-amino-acid exchanges within the virus-receptor binding site, which, however, come at the expense of viral fitness. IMPORTANCE The emergence of resistant viruses is one of the most frequent obstacles preventing successful therapy of viral infections, representing a significant threat to human health. We investigated the emergence of resistant viruses during treatment with sCAR-Fc, a well-studied, highly effective antiviral molecule against CVB infections. Our data show the molecular aspects of resistant CVB3 mutants that arise during repetitive sCAR-Fc usage. However, drug resistance comes at the price of lower viral fitness. These results extend our knowledge of the development of resistance by coxsackieviruses and indicate potential limitations of antiviral therapy using soluble receptor molecules., (Copyright © 2020 Pinkert et al.)

Replicative ability of 5 oncolytic enterovirus strains was evaluated on a panel of 18 human normal and tumor cells. The capacity of each cell line to support replication of enterovirus strains varied. Cell lines weakly replicating one virus could be highly sensitive to another viral strain. Differences in the expression of CXADR cell receptor did not correlate with susceptibility to infection and replication of Coxsackie B virus, but complete inactivation of CXADR gene and poliovirus receptor gene (PVR) led to loss of the sensitivity to Coxsackie B5 and poliovirus, respectively. Detection of additional expression markers will contribute to understanding the causes of different sensitivity of tumor cells to viruses.

The coxsackievirus and adenovirus receptor (CAR) has been studied extensively since its identification and isolation in 1997. The CAR is an immunoglobulin superfamily protein with two extracellular Ig-like domains, a single membrane-spanning sequence, and a significant cytoplasmic domain. It is structurally and functionally similar to the junctional adhesion molecules. The amino terminal domain, distal from the membrane, has been structurally characterized alone, bound to the adenovirus fiber knob, and, in full-length CAR, docked in the canyon structure of the coxsackievirus capsid. Although the past decade has produced a burst of new knowledge about CAR, significant questions concerning its function in normal physiology and coxsackievirus-related pathology remain unanswered.

The review presents the currently available data on the receptor specificity of enteroviruses. It discusses whether changes in the receptor specificity of enteroviruses may play a role in their in vitro and in vivo reproduction.

Many entero-, parecho-, and rhinoviruses use immunoglobulin (Ig)-like receptors that bind into the viral canyon and are required to initiate viral uncoating during infection. However, some of these viruses use an alternative or additional receptor that binds outside the canyon. Both the coxsackievirus-adenovirus receptor (CAR), an Ig-like molecule that binds into the viral canyon, and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). A cryoelectron microscopy reconstruction of a variant of CVB3 complexed with DAF shows full occupancy of the DAF receptor in each of 60 binding sites. The DAF molecule bridges the canyon, blocking the CAR binding site and causing the two receptors to compete with one another. The binding site of DAF on CVB3 differs from the binding site of DAF on the surface of echoviruses, suggesting independent evolutionary processes.

Enteroviruses, such as the coxsackievirus (CV) group, have been linked to the induction of inflammatory and autoimmune diseases. Virus tropism and tissue access are modulated by endothelial cells. To examine the susceptibility of microvascular endothelial cells (MECs) derived from pancreatic islets to infection with CV group B (CVB), purified cultured human islet MECs were infected with CVB-4 strain, and the immunological phenotype of the infected cells was analyzed. CVB-4 persistently infected the islet MECs, which expressed the CV receptors human coxsackievirus and adenovirus receptor (HCAR) and decay accelerating factor (DAF) and maintained EC characteristics, without overt cytopathic effects. CVB-4 infection transiently up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1 and increased production of the proinflammatory cytokines IL-1beta and IL-6, and chemokines IL-8 and lymphotactin, as well as IFN-alpha. Mononuclear cell adhesion to CVB infected monolayers was increased, compared to uninfected monolayers. Moreover, infection up-regulated the viral receptors HCAR and DAF and coreceptor alpha(v)beta3 integrin on islet MECs, while down-regulating expression of HCAR on human aortic endothelial cells, indicating potential tissue-specific influence on the pathological outcome of infection. These results provide evidence that islet MECs are natural targets and reservoirs for persistent CVB infection resulting in acute endothelial cell activation by virus, which may contribute to selective recruitment of subsets of leukocytes during inflammatory immune responses, such as insulitis in type 1 diabetes.

Swine vesicular disease virus (SVDV) evolved from coxsackie B virus serotype 5 (CVB5) in the recent past, crossing the species barrier from humans to pigs. Here, SVDV isolates from early and recent outbreaks have been compared for their capacity to utilize the progenitor virus receptors coxsackie-adenovirus receptor (CAR) and decay-accelerating factor (DAF; CD55). Virus titre of CVB5 and SVDV isolates It'66 and UK'72 on human HeLa cells was reduced by pre-incubation with either anti-DAF or anti-CAR antibodies; however, recent SVDV isolates R1072, R1120 and SPA'93 did not infect HeLa cells lytically. CVB5 and SVDV infection of the pig cell line IB-RS-2 was inhibited completely by anti-CAR antibodies for all isolates, and no reduction was observed following pre-incubation of cells with anti-pig DAF antibodies. Expression of human DAF in the pig cell line IB-RS-2 enhanced the virus titre of early SVDV isolates by 25-fold, but had no effect on recent SVDV isolate titre. Binding of radiolabelled CVB5 to IB-RS-2 cells was increased seven- to eightfold by expression of human DAF and binding of early SVDV isolates was increased 1.2-1.3-fold, whereas no increase in binding by recent SVDV isolates was mediated by human DAF expression. Addition of soluble hDAF-Fc inhibited CVB5, but not SVDV, infection of pig cells. Pre-incubation of all viruses with soluble hCAR-Fc blocked infection of IB-RS-2 pig cells completely; titration of the amount of soluble hCAR-Fc required to block infection revealed that early isolate UK'72 was the least susceptible to inhibition, and the most recent isolate, SPA'93, was the most susceptible.

Although many coxsackie B viruses interact with decay accelerating factor (DAF), attachment to DAF by itself is not sufficient to initiate infection. We examined the early events in infection that follow virus interaction with DAF, and with the coxsackievirus and adenovirus receptor (CAR). Interaction with soluble CAR in a cell-free system, or with CAR on the surfaces of transfected cells, induced the formation of A particles; interaction with soluble or cell surface DAF did not. The results suggest that CAR, but not DAF, is capable of initiating the conformational changes in the viral capsid that lead to release of viral nucleic acid.

Coxsackievirus A9 (CAV9), a member of the Enterovirus genus of Picornaviridae, is a common human pathogen and is one of a significant number of viruses containing a functional arginine-glycine-aspartic acid (RGD) motif in one of their capsid proteins. Previous studies identified the RGD-recognizing integrin alpha(v)beta(3) as its cellular receptor. However, integrin alpha(v)beta(6) has been shown to be an efficient receptor for another RGD-containing picornavirus, foot-and-mouth disease virus (FMDV). In view of the similarity in sequence context of the RGD motifs in CAV9 and FMDV, we investigated whether alpha(v)beta(6) can also serve as a receptor for CAV9. We found that CAV9 can bind to purified alpha(v)beta(6) and also to SW480 cells transfected with beta(6) cDNA, allowing expression of alpha(v)beta(6) on their surface, but it cannot bind to mock-transfected cells. In addition, a higher yield of CAV9 was obtained in beta(6)-expressing cells than in mock-transfected cells. There was no similar enhancement in infection with an RGD-less CAV9 mutant. We also found beta(6) on the surface of GMK cells, a cell line which CAV9 infects efficiently by an RGD-dependent mechanism. Significantly, this infection is blocked by an antibody to alpha(v)beta(6), while this antibody did not block the low level of infection by the RGD-less mutant. Thus, integrin alpha(v)beta(6) is an RGD-dependent receptor for CAV9 and may be important in natural CAV9 infections.

The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus B (CVB) and adenovirus (Ad). The normal cellular function of CAR, which is expressed in a wide variety of tissue types, is thought to involve homophilic cell adhesion in the developing brain. The extracellular domain of CAR consists of two immunoglobulin (Ig) domains termed CAR-D1 and CAR-D2. CAR-D1 is shown by sedimentation velocity to be monomeric at pH 3.0. The solution structure and the dynamic properties of monomeric CAR-D1 have been determined by NMR spectroscopy at pH 3.0. The determinants of the CAR-D1 monomer-dimer equilibrium, as well as the binding site of CVB and Ad on CAR, are discussed in light of the monomer structure.

In this study, a zebrafish homologue of the coxsackievirus and adenovirus receptor (CAR) protein was identified. Although the extracellular domain of zebrafish CAR (zCAR) is less than 50% identical to that of human CAR (hCAR), zCAR mediated infection of transfected cells by both adenovirus type 5 and coxsackievirus B3. CAR residues interacting deep within the coxsackievirus canyon are highly conserved in zCAR and hCAR, which is consistent with the idea that receptor contacts within the canyon are responsible for coxsackievirus attachment. In contrast, CAR residues contacting the south edge of the canyon are not conserved, suggesting that receptor interaction with the viral "puff region" is not essential for attachment.

We have used X-ray crystallography to determine the structure of a decay accelerating factor (DAF)-binding, clinic-derived isolate of echovirus 11 (EV11-207). The structures of the capsid proteins closely resemble those of capsid proteins of other picornaviruses. The structure allows us to interpret a series of amino acid changes produced by passaging EV11-207 in different cell lines as highlighting the locations of multiple receptor-binding sites on the virion surface. We suggest that a DAF-binding site is located at the fivefold axes of the virion, while the binding site for a distinct but as yet unidentified receptor is located within the canyon surrounding the virion fivefold axes.

Many picornaviruses use cell-surface molecules belonging to the immunoglobulin superfamily (IgSF) as their cellular receptors. These molecules usually consist of tandem repeats of between two and five Ig-like domains whose amino-terminal domains (D1) interact with invading viruses, with their carboxy-terminal sections comprising a transmembrane and a short cytoplasmic region. Most rhino- and enteroviruses, belonging to the Picornavirus family, use a canyon-like feature on their surface to attach to cellular receptors. Binding into the canyon destabilizes the virus and thus initiates the uncoating process. By contrast, non-IgSF molecules, when used by picornaviruses as receptors, bind outside the canyon and do not cause viral instability.

Strain-specific differences in the interaction of coxsackievirus B3 (CVB3) with the coxsackievirus-adenovirus receptor (CAR) and the decay-accelerating factor (DAF) co-receptor proteins were investigated using a non-haemagglutinating (CVB3) and a haemagglutinating (CVB3-HA) strain of CVB3. A panel of receptor-transfected hamster CHO cells, expressing either CAR (CHOCAR cells), DAF (CHODAF cells), or both receptor proteins (CHODC cells) were used to study the interplay of CAR and DAF receptor molecules with regard to binding and infection with CVB3 and CVB3-HA. Despite clear differences in their binding phenotypes, both virus strains were found to primarily depend on the CAR receptor protein for initialization of productive infections. Cytopathic effects induced by CVB3-HA were influenced by co-expression of DAF receptor proteins. The cardiotropic potential of both virus strains was investigated in A.BY/SnJ mice. Despite comparable virus replication of both CVB3 strains in individual myocytes, the number of infected heart muscle cells was significantly lower in CVB3-HA infected mice. Infections of pancreata correlated with myocardial infections. Together these data suggest that even small differences in virus-receptor interactions, influencing virus binding and virus spread, may have an impact on the pathogenesis of CVB-induced diseases., (Copyright 2002 Wiley-Liss, Inc.)

Coxsackie B viruses (CVB) are enteroviruses belonging to the family Picornaviridae. Serotypes 1, 3 and 5 of CVB bind to the human membrane complement regulator decay-accelerating factor (DAF) and the coxsackievirus/adenovirus receptor (CAR), using either or both as receptors. These viruses are known to infect pig cell lines, but the receptor(s) involved has not been identified. We have recently characterized the pig homologue of DAF and here explore the interactions of human DAF-binding CVB with pig homologues of DAF and CAR. CVB infection of three pig cell lines resulted in cytolysis, which could be not be blocked by anti-pig DAF antibodies. CVB bound to CHO cells transfected with human DAF, but not pig DAF. Modification of pig DAF by incorporation of the fourth short consensus repeat of human DAF did not confer CVB-binding capacity. CVB did bind CHO cells expressing pig or human CAR, and pre-incubation of pig cells with anti-CAR antibody blocked CVB infection.

The coxsackievirus and adenovirus receptor protein (CAR) serves as the cell surface receptor for group B coxsackieviruses and most adenoviruses, but the physiological function and ligand for this protein remain to be described. An affinity column was constructed with the recombinant extracellular domain of the CAR (rECAR) to isolate potential ligands by affinity chromatography. Immunoglobulins G and M were consistently isolated from human sera passed through the column, suggesting that the CAR may be an immunoglobulin-binding protein. Further investigation revealed that the affinity-purified immunoglobulins bound to rECAR-coated immunoassay plates, and the peroxidase-labeled rECAR bound the immunoglobulins on ligand-overlay blots. The peroxidase-labeled rECAR was incorporated into immunoprecipitates formed between the affinity-purified immunoglobulins and rabbit antibodies against human immunoglobulins, but not into immunoprecipitates formed between mouse IgG and rabbit antibodies against mouse IgG. The CAR present in HeLa cell lysates also bound to the affinity-purified immunoglobulins on Immobilon membranes, showing that the association is not limited to the recombinant protein. These results demonstrate that the CAR binds IgG and IgM present in serum, and reveal a direct interaction between the coxsackievirus and adenovirus receptor and the immune system.

Group B coxsackieviruses (CVB) utilize the coxsackievirus-adenovirus receptor (CAR) to recognize host cells. CAR is a membrane protein with two Ig-like extracellular domains (D1 and D2), a transmembrane domain and a cytoplasmic domain. The three-dimensional structure of coxsackievirus B3 (CVB3) in complex with full length human CAR and also with the D1D2 fragment of CAR were determined to approximately 22 A resolution using cryo-electron microscopy (cryo-EM). Pairs of transmembrane domains of CAR associate with each other in a detergent cloud that mimics a cellular plasma membrane. This is the first view of a virus-receptor interaction at this resolution that includes the transmembrane and cytoplasmic portion of the receptor. CAR binds with the distal end of domain D1 in the canyon of CVB3, similar to how other receptor molecules bind to entero- and rhinoviruses. The previously described interface of CAR with the adenovirus knob protein utilizes a side surface of D1.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

Some echoviruses (EV) that bind decay-accelerating factor (DAF) also bind cells of human and murine origins in a DAF-independent manner. Pretreatment of cells with heparinase 1 or heparin blocks the binding of radiolabeled virus to the cell surface, and heparin prevents infection of rhabdomyosarcoma cells by certain EV, including several low-passage clinical isolates of EV 6 and some EV that do not bind DAF. These studies suggest that heparan sulfate may be of in vivo relevance as an attachment molecule for EV.

Background: Virus-receptor binding is an essential step in every virus infectious process. Many viruses employ more than one receptor molecule or even receptor complexes for attachment. In this study, we investigate the binding of Echovirus 1 (Echo1) and Coxsackievirus A9 (CAV-9) on cell surface molecules. CAV-9 has been reported to utilize integrin alpha v beta(3) in binding to cells, whereas Echo1 has been known to utilize integrin alpha 2 beta(1). METHODS AND RESULTS We directly test whether the presence of these molecules alone was sufficient for virus binding. We devised a novel flow cytometric binding assay that enables us to quantify virus particles bound on host cells and to further determine the extent to which viruses utilize specific receptors., Conclusions: By quantifying virus particles and possible receptor molecules, we found that Echo1 utilizes mainly integrin alpha 2 beta(1). CAV-9 utilizes integrin alpha v beta(3) to a much lesser extent (40%), indicating that CAV-9 also utilizes other receptor(s)., (Copyright 2001 Wiley-Liss, Inc.)

Viruses that propagate within vertebrate hosts have adapted many strategies to infect host cells. One of the first steps in a viral infection is the binding of the virus to cell surface molecules. This interaction between a virus and its receptors plays a key role in the multiplication cycle. Entry of viruses into cells is a complex, multistep process, and for several viruses, cell attachment and internalization are distinct steps. A number of virus receptors have been identified; a common family of viral receptors is the integrin family. Integrins are a widely expressed family of cell adhesion receptors, by which cells attach to extracellular matrices; they also mediate important cell-cell adhesion events. Integrins are involved in a number of tissue remodeling events, including embryogenesis, angiogenesis, wound repair, and bone resorption. In addition, several integrins are used by manyviruses in theirinfectious cycle. Virus-integrin interactions maybe more complex than previously thought because several viruses can interact with unique integrin regions or can activate distinct signaling pathways. This article will discuss the strategies devised by many viruses in their integrin-mediated attachment or cell entry.

Trypsin treatment of HeLa cells results in a limited proteolysis of the coxsackievirus and adenovirus receptor (CAR) after which the cleaved CAR remains cell-associated and tryptic peptides remain associated through disulfide bonds. Trypsin-treated HeLa cells remain susceptible to infection with coxsackievirus B and produce progeny virus at 8 h post-infection in amounts comparable to cells with intact CAR. HeLa cells remove the proteolysed CAR within 15 h and require over 24 h to restore intact CAR to control levels. As turnover is relatively slow, physiological functions that require intact CAR protein may be compromised for more than 24 h following trypsin treatment. Moreover, since removal of proteolysed CAR proceeds at more than twice the replacement rate, trypsin treatment disrupts the receptor-per-cell steady state for at least 24 h.

Selected members of the adenovirus family have been shown to interact with the coxsackie adenovirus receptor, alpha(v) integrins, and sialic acid on target cells. Initial interactions of subgenus D adenoviruses with target cells have until now been poorly characterized. Here, we demonstrate that adenovirus type 8 (Ad8), Ad19a, and Ad37 use sialic acid as a functional cellular receptor, whereas the Ad9 and Ad19 prototypes do not.

Coxsackievirus type B (CVB) infection of the pancreas induces a massive cellular infiltrate composed of natural killer cells, T cells, and macrophages and leads to the destruction of exocrine tissue. The physiological manifestations of pancreatic CVB infection are correlated with viral tropism; the virus infects acinar cells but spares the islets of Langerhans. Here we evaluate the mechanisms underlying pancreatic inflammation and destruction and identify the determinants of viral tropism. T-cell-mediated immunopathology has been invoked, along with direct virus-mediated cytopathicity, to explain certain aspects of CVB-induced pancreatic disease. However, we show here that in the pancreas, the extent of inflammation and tissue destruction appears unaltered in the absence of the cytolytic protein perforin; these findings exclude any requirement for perforin-mediated lysis by natural killer cells or cytotoxic T cells in CVB3-induced pancreatic damage. Furthermore, perforin-mediated cytotoxic T-cell activity does not contribute to the control of CVB infection in this organ. In addition, we demonstrate that the recently identified coxsackie-adenovirus receptor is expressed at high levels in acinar cells but is barely detectable in islets, which is consistent with its being a major determinant of virus tropism and, therefore, of disease. However, further studies using various cell lines of pancreatic origin reveal secondary determinants of virus tropism., (Copyright 2000 Academic Press.)

The coxsackievirus group B (CVB) and adenovirus (Ad) receptor (HCVADR, formerly HCAR) is a cell surface protein with two immunoglobulin-like regions (IG1 and IG2) that serves as a receptor for two structurally unrelated viruses. We have established the tissue distribution of the receptor in the rodent by immunohistochemistry and show that the receptor is broadly expressed during embryonic development in the central and peripheral nervous systems and in several types of epithelial cells. The tissue distribution is more restricted in the adult but remains high mainly in epithelial cells. Using site-directed mutagenesis, based on computer modeling of the IG1 region, Ad5 binding could be inhibited but CVB attachment was unaffected. A double amino acid substitution in a three-stranded anti-parallel beta sheet that may form a face of the receptor completely inhibited Ad5 binding. Therefore, we conclude that the molecular interactions critical for Ad5 binding to HCVADR do not overlap with those of CVB3. In fact a specific antibody interfering with only CVB binding recognizes the IG2 domain in the receptor, suggesting that the CVB interacts with this region or an overlap between the IG1 and the IG2 regions., (Copyright 2000 Academic Press.)

Many serotypes of echovirus (EV) and Coxsackie B virus (CBV) bind human decay-accelerating factor (DAF) and use it as a receptor for infection. Analogues for DAF have been isolated from mice and rats and characterized; these analogues have amino acid identities to human DAF of approximately 60%. EV serotypes 3, 6', 7, 11-13, and 29 and CBV serotypes 1, 3, and 5 caused hemagglutination of human erythrocytes but not rat or mouse erythrocytes, suggesting failure to bind rodent DAF. To confirm this evidence, radiolabeled viruses were incubated with transfected Chinese hamster ovary (CHO) cells that were abundantly expressing each type of DAF. Only cells that expressed human DAF bound virus. Although binding of EV and CBV was specific for human DAF, complement inhibition by DAF expressed in CHO cells was similar for each analogue.

Decay-accelerating factor (DAF/CD55), and coxsackievirus-adenovirus receptor (CAR) have been identified as cellular receptors for coxsackie B viruses (CBV). To elucidate the interplay of DAF and CAR on the cell surface, virus-receptor interactions of two coxsackieviruses of serotype B3 (non-haemagglutinating CBV3 and haemagglutinating CBV3-HA strain) were analysed. Binding assays revealed clear differences between these viruses with regard to their interactions with DAF and CAR. However, only the combination of anti-DAF and anti-CAR antibodies resulted in complete inhibition of virus binding for both strains. In plaque-reduction assays, anti-DAF antibodies had no effect, whereas CAR-specific antibodies significantly reduced productive infection of HeLa cells by both viruses. Interestingly, a synergistic inhibitory effect of anti-DAF and anti-CAR antibodies was also observed with regard to infection. These findings support the model of preferential interactions of both strains of CBV3 with closely associated DAF and CAR proteins on HeLa cells, despite displaying clear differences in their binding phenotypes.

Therapeutic interference with virus-cell surface receptor interactions represents a viable antiviral strategy. Here we demonstrate that cytoplasmic expression of the serine protease inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), affords a high level of protection from lytic infection by multiple human picornaviruses. The antiviral action of PAI-2 was mediated primarily through transcriptional down-regulation of the following virus receptors: intercellular adhesion molecule 1 (ICAM-1, a cellular receptor for the major group of rhinoviruses), decay-accelerating factor (a cellular receptor for echoviruses and coxsackieviruses), and to a lesser extent the coxsackie-adenovirus receptor protein (a cellular receptor for group B coxsackieviruses and group C adenoviruses). Expression of related cell surface receptors, including membrane cofactor protein and the poliovirus receptor, remained unaffected. These findings suggest that PAI-2 and/or related serpins may form the basis of novel antiviral strategies against picornavirus infections and also therapeutic interventions against ICAM-1-mediated respiratory inflammation.

The extracellular region of the coxsackievirus and adenovirus receptor (CAR) is predicted to consist of two immunoglobulin (Ig)-related structural domains. We expressed the isolated CAR amino-terminal domain (D1) and a CAR fragment containing both extracellular Ig domains (D1/D2) in Escherichia coli. Both D1 and D1/D2 formed complexes in vitro with the recombinant knob domain of adenovirus type 12 (Ad12) fiber, and D1 inhibited adenovirus type 2 (Ad2) infection of HeLa cells. These results indicate that the adenovirus-binding activity of CAR is localized in the amino-terminal IgV-related domain and confirm our earlier observation that Ad2 and Ad12 bind to the same cellular receptor. Preliminary crystallization studies suggest that complexes of Ad12 knob bound to D1 will be suitable for structure determination.

The biochemical properties of the molecular interactions mediating viral-cell recognition are poorly characterized. In this study, we use surface plasmon resonance to study the affinity and kinetics of the interaction of echovirus 11 with its cellular receptor decay-accelerating factor (CD55). As reported for interactions between cell-cell recognition molecules, the interaction has a low affinity (KD approximately 3.0 microM) as a result of a very fast dissociation rate constant (kon approximately 10(5) M-1.s-1, koff approximately 0.3 s-1). This contrasts with the interaction of soluble ICAM-1 (sICAM-1, CD54) with human rhinovirus 3 which has been reported to have a similar affinity but 10(2)-10(3)-fold slower kinetics (Casasnovas, J. M., and Springer, T. A. (1995) J. Biol. Chem. 270, 13216-13224). The extracellular portion of decay-accelerating factor comprises four short consensus repeat domains (domains 1-4) and a mucin-like stalk. By comparison of the binding affinity for echovirus 11 of various fragments of decay-accelerating factor, we are able to conclude that short consensus repeat domain 3 contributes approximately 80% of the binding energy.

Several echoviruses (EVs) have previously been shown to use decay accelerating factor (DAF) as a cellular receptor. Since DAF is expressed on erythrocytes, EVs that use this receptor cause haemagglutination. Here we show that all EVs that haemagglutinate do so via attachment to DAF and that this interaction can be inhibited by a monoclonal antibody (MAb) specific for DAF domain SCR3. Although the viruses haemagglutinate via DAF some can bind to rhabdomyosarcoma cells from which DAF has been removed and infect in the presence of a MAb against DAF. This suggests that some EVs have the capacity to interact with more than one cellular receptor.

The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.

Soluble forms of decay-accelerating factor (DAF) (CD55), the receptor for echovirus 7, were synthesized in the yeast Pichia pastoris. Purified recombinant protein containing SCR domains 2, 3, and 4, but lacking the serine/threonine rich region, was shown to block infection of susceptible cells by echovirus 7. In contrast to the situation with poliovirus and its receptor, the neutralization of echovirus 7 by soluble DAF was completely reversible and did not lead to the formation of 135S A-particles. Binding of virus to susceptible cells, by contrast, did lead to the formation of A particles, mainly from virus that had been internalized. The data suggest that a secondary factor(s) may contribute to A-particle formation and uncoating of echovirus 7.

The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1. Binding sites for both collagen and echovirus 1 have been mapped to the I domain within the alpha2 subunit of the VLA-2 alpha2beta1 heterodimer. Although murine VLA-2 interacts with collagen, it does not bind virus. We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199-201 and 212-216, independently involved in virus attachment. These residues are distinct from the metal ion-dependent adhesion site previously demonstrated to be essential for VLA-2 interactions with collagen. Mutations in three metal ion-dependent adhesion site residues that abolish adhesion to collagen had no effect on virus binding. These results confirm that different sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands.

Viral infection is a complex process which includes binding and interaction of the virus with specific cell surface receptors, uptake and uncoating of the virus, and finally replication. Chinese hamster ovary (CHO) cells are non-permissive towards infection with coxsackieviruses of group B (CVB), although they do express a putative CVB-specific receptor protein. In order to localize the block of infection in CHO cells, these cells were tested for binding of radiolabelled CVB3, receptor-mediated transformation of virions into A-particles, and replication of the viral genome. Binding of CVB3 to CHO cells was found to be comparable to the binding of this virus to permissive cell lines. Detergent-solubilized membrane proteins of CHO cells were tested in virus overlay protein-binding assays (VOPBAs) and shown to express a 100 kDa CVB-binding membrane protein similar to the CVB receptor protein which we recently described for permissive HeLa cells. Incubation of CVB3 with intact CHO cells resulted in transformation of cell-bound virions into non-infectious A-particles (deprived of capsid protein VP4), demonstrating the functional activity of the CVB receptor protein on CHO hamster cells. Transfection of recombinant CVB3 cDNA or viral RNA into CHO cells resulted in the production of infectious CVB3 virions, implying that the failure of CVB to infect CHO cells is not caused by a defect in virus replication but results from a block in the uptake of virus particles into the cell after the initial steps of virus-receptor interactions.

Coxsackie B viruses interact with two putative cell surface receptor molecules. Experiments with prototype laboratory strains suggest that all 6 coxsackie B serotypes interact with a 46-kDa protein recognized by the monoclonal antibody RmcB, whereas CB1, CB3, and CB5 may also bind to decay accelerating factor. Antireceptor monoclonal antibodies were used to study interactions between low-passage clinical coxsackie B virus isolates and the two receptors. In contrast to observations made with single prototype strains, these data indicate that receptor use by clinical isolates is not strictly related to serotype and that even prototype strains with different passage histories may differ in receptor use. Within a given serotype, variation exists in the capacity of individual virus isolates to bind to specific receptors, and variants with altered receptor specificity may arise during infection in humans and in tissue culture.

A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection. Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery with adenovirus vectors.

This study was designed mainly to determine the relationships between the expression and distribution of the cellular receptor proteins for coxsackievirus B3 (CVB3) and susceptibility of mouse brain cells during fetal development of Balb/c mice. Immunoblot analysis of fetal extracts demonstrated that the CVB3 receptor proteins were first expressed at day 14 of the fetal stage, and that maximal expression of the cellular receptor occurred at near term or newborn stage. Results also suggested that newborn mouse brain tissue expressed much larger quantities of viral receptor proteins, compared to other tissues. In vitro studies showed that both mouse neurons and astrocytes could be infected by two CVB3 strains, pantropic CVB3 Nancy strain (CVB3N) and myocardiotropic CVB3 Woodruff strain (CVB3W). CVB3N, however, replicated and grew to high titer in primary astrocyte cultures and in primary neuron cultures, whereas, primary astrocyte cultures were relatively resistant to CVB3W. Virus binding assays revealed that CVB3N bound faster and in greater amounts to mouse brain cells than CVBW. These two virus strains, however, were found to share the same receptor specificity by virus competition assays. The number of virus binding sites for CVB3 on newborn mouse brain cells was approximately 1.8 x 10(4) per cell. The data suggested that preferential expression of the cellular receptors on newborn mouse brain cells may be related to their high susceptibilities to CVB3 infection.

Receptor binding and subsequent cell-mediated internalization or disassembly are the initial steps in virus replication. Cell surface molecules that participate in this process are the primary determinants of virus tissue tropism. Monoclonal antibody blockade, immunoprecipitation, and DNA transfection were used to identify decay accelerating factor as a major cell attachment receptor for coxsackieviruses B1, B3, and B5. However, expression of human decay acceleration factor on the surface of nonpermissive murine fibroblasts led only to virus attachment without subsequent replication, and it was concluded that an additional cellular cofactor(s) is required to facilitate cell entry and subsequent replication.

A coxsackievirus B3 (CB3) isolate adapted to growth in RD cells shows an alteration in cell tropism as a result of its capacity to bind a 70-kDa cell surface molecule expressed on these cells. We now show that this molecule is the complement regulatory protein, decay-accelerating factor (DAF) (CD55). Anti-DAF antibodies prevented CB3 attachment to the cell surface. Radiolabeled CB3 adapted to growth in RD cells bound to CHO cells transfected with human DAF, whereas CB3 (strain Nancy), the parental strain, did not bind to DAF transfectants. These results indicate that growth of CB3 in RD cells selected for a virus strain that uses DAF for cell surface attachment.

The integrin VLA-2 mediates cell adhesion to collagen and laminin and also functions as a virus receptor, mediating cell surface attachment and infection by a human pathogen, echovirus 1. To determine whether extracellular matrix proteins and virus interact with VLA-2 in the same manner, we carried out a detailed comparison of these two functions and found that they differed markedly in six different respects. In contrast to the ECM/VLA-2 interaction, echovirus 1 binding did not discriminate between functional forms of VLA-2, showed a different pattern of inhibition by anti-beta1 and -alpha 2 antibodies, was not stimulated by phorbol esters, was not activated by beta 1 antibodies that stimulate ECM binding, was not inhibited by any particular divalent cation, and most notably was not inhibited by EDTA. These striking differences were found both with intact cells expressing VLA-2 and with solubilized VLA-2, suggesting that VLA-2 interacts with these different ligands by markedly different mechanisms, and probably at different functional sites. In addition, alterations in the alpha 2 cytoplasmic domain that had marked effects on cellular responses to collagen and laminin had no effect on virus internalization and cell killing. Thus VLA-2-mediated events that occur after receptor occupancy by extracellular matrix proteins also appear to be distinct from those that occur after receptor interaction with virus.

We previously reported that infection of KB cells by echoviruses (EV) was inhibited by a KB-derived EV receptor murine monoclonal antibody (mAb 143). This antibody enabled the identification of a cellular receptor common to all echoviruses (with the exception of EV-22 and -23) and coxsackievirus (CV) A9, but different from the receptor of other picornaviruses. We now present results of cell protection assays conducted with human and simian cell lines different from the KB cell line used for production of mAb 143. When human embryonic lung fibroblasts were pretreated with 150 micrograms/ml of mAb 143, EV-11 and CV-A9 were completely inhibited (more than a 2-log difference compared to untreated cells). When the cell protection experiments were performed with Vero cells, the same results were observed with EV-33, but not with EV-22. The protection afforded human fibroblast cells by mAb 143 persisted for at least 5 days after 2-h exposure to 100 TCID50 of EV-11. These results suggest that EV receptors can be effectively blocked for prolonged periods in susceptible cells.

Cell surface receptors for echovirus, a common human pathogen, were identified with monoclonal antibodies that protected susceptible cells from infection with echovirus 1. These monoclonal antibodies, which prevented virus attachment to specific receptor sites, recognized the alpha and beta subunits of the integrin VLA-2 (alpha 2 beta 1), a receptor for collagen and laminin. RD rhabdomyosarcoma cells expressed little VLA-2, did not bind to 35S-labeled virus, and resisted infection until transfected with complementary DNA encoding the alpha 2 subunit of VLA-2. Thus, integrins, adhesion receptors important in interactions between cells and with the extracellular matrix, can mediate virus attachment and infection.

Competition binding studies between viruses are usually performed with radiolabelled probes. In this report, a cytofluorimetric method using biotinylated echovirus (EV) 11 is described for the study of competition of enteroviruses for a common cell receptor site. An N-hydroxysuccinimide ester biotin spacer arm was used for biotinylation of CsSO4-purified EV 11. Biotinylation did not change the infectivity of the virus (attachment to and replication in susceptible cells). With the exception of EV 22 and EV 23, all the echovirus serotypes and also coxsackievirus A9 (CA 9) were able to inhibit the absorption of biotinylated EV 11 onto cells. The taxonomic implications of these findings are discussed.

The properties of solubilized receptors for coxsackieviruses B (CBV) on HeLa cells were investigated. It was found that the receptor activity and antigenicity were comparatively sensitive to pH change and were rather stable over a wide range of temperatures (4 degrees C-60 degrees C) but they were destroyed completely when the temperature rose to 100 degrees C. Oxidation and reduction augmented the receptor antigenicity, while the receptor activity was lowered only by oxidation and not by reduction. After lipoprotein was precipitated with heparin buffer, the receptor activity and antigenicity remained in the supernatant. Treatment of the solubilized receptors with proteinase K, trypsin and elastase could partially or completely inactivate the viral receptors. The results suggest that the receptors for CBV on HeLa cells are glycoproteins insensitive to temperature and their sulfhydryl groups are unnecessary for receptor activity.

The selective proton NMR relaxation rates were measured in the system made by the local anesthetic procaine, human erythrocytes and the hemagglutinating Echo virus type 11. The NMR technique provided a very good tool for using drugs firmly bound to receptor membranes as biological "markers" of the virus-cell interaction.

Physical and chemical properties of deoxycholate-solubilized HeLa cell plasma membrane receptors for binding group B coxsackieviruses were determined. Receptors eluted from Sepharose 4B with an apparent molecular weight of 275,000 and sedimented with an S value of between 14.7 and 4.9 and a buoyant density of 1.06 to 1.10 g/cm3. Virus-binding activity was destroyed after treatment with proteases, glycosidases, and periodate but was unaffected by lipases or reducing or alkylating agents. Additionally, lectins, including concanavalin A, adsorbed receptors and inhibited virus attachment. The composite data suggested that glycoprotein is an integral part of the receptors for binding virus.

Enterovirus type 70 (EV70) agglutinated human 'O' erythrocytes at 4 degrees C as well as 22 degrees C, but visible agglutination was lost when warmed at 37 degrees C although the virus remained attached to the surface of the erythrocyte. The receptor sites for the virus were neuraminidase-sensitive. A direct involvement of sialic acid on the cell surface in virus-cell interaction was confirmed by the fact that the presence of fetuin or free N-acetylneuraminic acid inhibited the haemagglutinating activity of EV70. Similar numbers of virus particles were required for 1 haemagglutinating unit (HAU) of EV70 and 1 HAU of mengovirus, whereas 2.6-fold or more of virus particles of echovirus type 7 and type 11 gave the same activity. On the other hand, the number of receptor sites on the cell surface for EV70 was found to be sevenfold more than for mengovirus. Therefore, the erythrocyte receptor for EV70 is different from that for common enteroviruses and similar, though not identical, to the cardiovirus receptor. However, serological tests such as neutralization, complement fixation or haemagglutination inhibition did not reveal any common antigen between EV70 and cardiovirus.