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R Abu-Ghazaleh

Publications and source records attributed to R Abu-Ghazaleh.

13 recordsLinked to original sources
PubMed1999

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

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

Adaptation, Physiological↗
PubMed1997

Vascular endothelial growth factor stimulates prostacyclin production and activation of cytosolic phospholipase A2 in endothelial cells via p42/p44 mitogen-activated protein kinase.

Vascular endothelial growth factor (VEGF) stimulated a time- and concentration-dependent increase in PGI2 synthesis in human umbilical vein endothelial cells with a mean maximum increase of 2-fold above basal levels at 25 ng/ml after 60 min. VEGF also rapidly stimulated the release of arachidonic acid and phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). The VEGF-related factor, placenta growth factor (PIGF), had little effect on PGI2 synthesis, arachidonic acid release or cPLA2 activation. PD98059, a selective inhibitor of MAP kinase kinase, caused complete inhibition of VEGF-stimulated MAP kinase activity, PGI2 synthesis and cPLA2 gel retardation, but had no effect on VEGF-induced vWF secretion. These findings provide the first evidence that VEGF can stimulate PGI2 synthesis via cPLA2-mediated arachidonic acid release and indicate that VEGF stimulation of this biosynthetic pathway may occur, at least in part, via activation of p42/p44 MAP kinases.

Arachidonic Acid↗
PubMed1997

Dissecting the roles of VP0 cleavage and RNA packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus.

Empty capsids of foot-and-mouth disease virus (FMDV) type A22 Iraq 24/64, whose structure has been solved by X-ray crystallography, are unusual for picornaviruses since they contain VP2 and VP4, the cleavage products of the protein precursor VP0. Both the N terminus of VP1 and the C terminus of VP4, which pack together close to the icosahedral threefold symmetry axis where three pentamers associate, are more disordered in the empty capsid than they are in the RNA-containing virus. The ordering of these termini in the presence of RNA strengthens interactions within a single protomer and between protomers belonging to different pentamers. The disorder in the FMDV empty capsid forms a subset of that seen in the poliovirus empty capsid, which has VP0 intact. Thus, VP0 cleavage confers stability on the picornavirus capsid over and above that attributable to RNA encapsidation. In both FMDV and poliovirus empty capsids, the internal disordering uncovers a conserved histidine which has been proposed to be involved in the cleavage of VP0. A comparison of the putative active sites in FMDV and poliovirus suggests a structural explanation for the sequence specificity of the cleavage reaction.

Aphthovirus↗
PubMed1996

Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity.

BACKGROUND: Foot-and-mouth disease virus (FMDV) is an extremely infectious and antigenically diverse picornavirus of cloven-hoofed animals. Strains of the A22 subtype have been reported to change antigenically when adapted to different growth conditions. To investigate the structural basis of this phenomenon we have determined the structures of two variants of an A22 virus. RESULTS: The structures of monolayer- and suspension-cell-adapted A22 FMDV have been determined by X-ray crystallography. Picornaviruses comprise four capsid proteins, VP1-4. The major antigenic loop of the capsid protein VP1 is flexible in both variants of the A22 subtype but its overall disposition is distinct from that observed in other FMDV serotypes (O and C). A detailed structural comparison between A22 FMDV and a type O virus suggests that different conformations in a portion of the major antigenic loop of VP1 (the GH loop, which is also central to receptor attachment) result in distinct folds of the adjacent VP3 GH loop. Also, a single mutation (Glu82-->Gly) on the surface of VP2 in the suspension-cell-adapted virus appears to perturb the structure of the VP1 GH loop. CONCLUSION: The GH loop of VP1 is flexible in three serotypes of FMDV, suggesting that flexibility is important in both antigenic variability and structural communication with other regions of the virus capsid. Our results illustrate two instances of the propagation of structural perturbations across the virion surface: the change in the VP3 GH loop caused by the VP1 GH loop and the Glu82-->Gly change in VP2 which we believe perturbs the GH loop of VP1. In the latter case, the amplification of the sequence changes leads to differences, between the monolayer- and suspension-cell-adapted viruses, in host-cell interactions and antigenicity.

Antigens, Viral↗
PubMed1995

Structural comparison of two strains of foot-and-mouth disease virus subtype O1 and a laboratory antigenic variant, G67.

BACKGROUND: Foot-and-mouth disease viruses (FMDVs) are members of the picornavirus family and cause an economically important disease of cloven-hoofed animals. To understand the structural basis of antigenic variation in FMDV, we have determined the structures of two viruses closely related to strain O1BFS whose structure is known. RESULTS: The two new structure are, like O1BFS, both serotype O viruses. The first, O1 Kaüfbeuren (O1K), is a field isolate dating from an outbreak of FMD in Europe in the 1960s. The second, called G67, is a quadruple mutant of O1K, generated in the laboratory, that bears point mutations conferring resistance to neutralizing by monoclonal antibodies, specific for each of the four major antigenic sites defined previously. The availability of the three related virus structures permits a detailed analysis of the way amino acid substitutions influence antigenicity. Structural changes are seen to be limited, in general, to the substituted side chain. For example, the GH loop of VP1, a highly antigenic and mobile protuberance which becomes ordered only under reducing conditions, was essentially indistinguishable in the three viruses despite the accumulation of up to four changes within its 15-residue sequence. At one of the other antigenic sites, however, changes between the two field strains did perturb both side-chain and main-chain structures in the vicinity. CONCLUSIONS: The conservation of conformation of the GH loop of VP1 adds to the evidence implicating an integrin as the cellular receptor for FMDV, since this loop contains a conserved RGD (Arg-Gly-Asp) sequence structurally similar to the same tripeptide in some other integrin-binding proteins. Structural changes required for the virus to escape neutralization by monoclonal antibodies are generally small. The more extensive type of structural change exhibited by the field isolates probably reflects differing selective pressures operating in vivo and in vitro.

Amino Acid Sequence↗
PubMed1994

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

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

Amino Acid Sequence↗
PubMed1994

The structure of an immunodominant loop on foot and mouth disease virus, serotype O1, determined under reducing conditions.

Residues 136-159 of VPI of foot and mouth disease virus (FMDV) comprise the G-H loop of the protein and form a prominent feature on the surface of virus particles. This sequence contains an immunodominant neutralizing epitope, which can be mimicked with synthetic peptides, and includes an Arg, Gly, Asp motif which has been implicated in the binding of the virus to cellular receptors. Crystallographic analysis of native virus particles failed to resolve the structure of this region due to its disordered state. However, reduction of a disulphide bond between cysteine residues 134 of VP1 and 130 of VP2 caused the G-H loop to collapse onto the surface of the virus particle and allowed its conformation to be determined.

Amino Acid Sequence↗
PubMed1993

Structure of a major immunogenic site on foot-and-mouth disease virus.

Attachment of foot-and-mouth disease virus (FMDV) to its cellular receptor involves a long and highly antigenic loop containing the conserved sequence, Arg-Gly-Asp, a motif known to be a recognition element in many integrin-dependent cell adhesion processes. In our original crystal structure of FMDV the Arg-Gly-Asp-containing loop ('the loop'), located between beta-strands G and H of capsid protein VP1, was disordered and hence essentially invisible. We previously surmised that its disorder is enhanced by a disulphide bond linking the base of the loop (Cys 134) to Cys 130 of VP2 (ref. 8). We report here the crystal structure of the virus in which this disulphide is reduced. Reduced virus retains infectivity and serological experiments suggest that some of the loop's internal structure is conserved. But here its structure has become sufficiently ordered to allow us to describe an unambiguous conformation, which we relate to some key biological properties of the virus.

Aphthovirus↗
PubMed1992

Crystallization and preliminary X-ray analysis of three serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease viruses from serotypes O, A and C have been crystallized. The particular strains studied include O1K, A10(61), A22 Iraq 24/64, A24 Cruzeiro and C-S8c1. In addition, crystals have been grown of G67, a monoclonal antibody neutralization escape mutant derived from O1K, and of virus R100, recovered after the establishment of a persistent infection in baby hamster kidney cells with C-S8c1. Empty particles, capsids which lack the RNA genome, have also been crystallized for subtypes A22 Iraq 24/64 and A10(61). In almost all cases, crystals suitable for high resolution structure determination were obtained from (NH4)2SO4 or mixtures of polyethylene glycol and NH4Cl.

Aphthovirus↗
PubMed1992

Release of granule proteins from eosinophils cultured with IL-5.

Eosinophils isolated from normal individuals were cultured in the presence of human rIL-5 (hrIL-5) for up to 14 days, and the effects of this exposure were determined. First, the hrIL-5-cultured eosinophils were activated and degranulated more readily than freshly isolated eosinophils. For example, eosinophils cultured for 7 days with hrIL-5 released 30 and 10% of granule eosinophil-derived neurotoxin (EDN) when exposed to Sepharose 4B beads coupled to secretory IgA and IgG, respectively, whereas freshly isolated eosinophils released only 19 and 4%, respectively, of their EDN in response to the same stimuli. Degranulation of hrIL-5-cultured eosinophils was not augmented by further exposure to hrIL-5, whereas degranulation of freshly isolated cells to secretory IgA and IgG beads was increased by exposure to hrIL-5. Second, eosinophils cultured with hrIL-5 had prolonged viability in vitro. For example, after four days of culture with 50 U/ml of hrIL-5, 86% of eosinophils were viable compared to 12% in medium alone. Third, hrIL-5-cultured eosinophils became hypodense, and electron microscopy showed that they contained granules with core and matrix lucency and with evidence of granule fusion. Fourth, hrIL-5-cultured eosinophils spontaneously lost 30 to 60% of their EDN, eosinophil cationic protein, and eosinophil peroxidase and about 50% of their eosinophil granule major basic protein content compared to freshly isolated eosinophils, and all four of the granule proteins were released into the culture medium. Fifth, detailed studies of eosinophils cultured in hrIL-5 showed that 89 +/- 10% of the starting quantity of EDN could be recovered at 7 days. Whereas 99 +/- 1% of the EDN at day 0 was cell associated, by 7 days 60 +/- 9% was in the cell supernatants. Thus, hrIL-5 activates eosinophils, increases their viability, decreases their density, and their content of granule proteins and causes release of the granule proteins into culture fluids. The striking loss of granule proteins during culture with hrIL-5 may be an important mechanism for deposition of these cationic toxins in various diseases where IL-5 plays a role.

Blood Proteins↗
PubMed1991

Effect of steroids on immunoglobulin-induced eosinophil degranulation.

Because glucocorticoids are a mainstay in the treatment of asthma and other allergic diseases, we tested the effect of various steroid hormones on secretory IgA- and IgG-induced eosinophil degranulation in vitro. Human normodense eosinophils were purified by discontinuous Percoll density gradient, and hypodense eosinophils were obtained by culture of normodense cells with recombinant human interleukin (rIL)-5. Eosinophils were incubated with various steroids, including dexamethasone, hydrocortisone, methylprednisolone, estradiol, or dihydrotestosterone at concentrations from 10(-9) to 10(-4) mol/L. Sepharose 4B beads coupled to ovalbumin, secretory IgA, or IgG were added as targets of degranulation and incubated at 37 degrees C for 4 hours. In some experiments, rIL-5 was added to eosinophils before addition of beads to activate the cells. The release of eosinophil-derived neurotoxin was measured by radioimmunoassay as an index of degranulation. Dexamethasone (10(-9) to 10(-4) mol/L), hydrocortisone (10(-9) to 10(-4) mol/L), estradiol (10(-9) to 10(-7) mol/L), and dihydrotestosterone (10(-9) to 10(-4) mol/L) had no effect on normodense eosinophil degranulation. Methylprednisolone, 10(-5) mol/L, inhibited degranulation of normodense eosinophils up to 20%, whereas 10(-4) mol/L inhibited degranulation of hypodense eosinophils, up to 30%. Overall, no difference in inhibition by steroids was observed between normodense and hypodense eosinophils. rIL-5 enhanced immunoglobulin-induced eosinophil degranulation, but this effect of rIL-5 was not blocked by any of the steroids tested. These results suggest that eosinophil degranulation and rIL-5-mediated eosinophil activation are not direct targets of glucocorticoids and that the beneficial effects of glucocorticoids on allergic inflammation in vivo are not likely due to direct effects on eosinophil degranulation.

Cell Degranulation↗
PubMed1990

Regulatory effect of cytokines on eosinophil degranulation.

We tested the effects of different cytokines on IgA- and IgG-induced eosinophil degranulation in vitro to determine the potential interaction between eosinophils and mononuclear cells. Purified normodense eosinophils were incubated with cytokines (including rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, rIL-6, IFN-gamma, granulocyte-macrophage CSF stimulating factor (GM-CSF), and TNF) for 1 to 3 h after which Ig-coupled Sepharose 4B beads were added as targets and the mixtures were incubated with the eosinophils at 37 degrees C for 4 h. The Ig used were secretory IgA (sIgA), serum IgA and IgG, and myeloma IgA and IgG. The release of eosinophil-derived neurotoxin (EDN) was measured by RIA as an index of degranulation. rIL-5 was the most potent enhancer of Ig-induced degranulation and increased EDN release by 48% for sIgA and 136% for IgG. The effect of rIL-5 appeared as quickly as 15 min after incubation of eosinophils, sIgA beads and IL-5. GM-CSF and rIL-3 also enhanced Ig-induced EDN release but less potently than rIL-5. GM-CSF and rIL-5 by themselves induced a small but significant release of EDN from eosinophils in the absence of Ig-coated beads; rIL-3 did not. However, IFN-gamma suppressed sIgA-induced EDN release by 23%. The other cytokines did not have any effect on eosinophil degranulation. These results suggest that cytokines which induce eosinophil differentiation and proliferation during hematopoiesis also enhance the effector function of mature eosinophils and that IFN-gamma partially down-regulates eosinophil degranulation.

Colony-Stimulating Factors↗