Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.

Persistent infection of K562 cells by encephalomyocarditis virus.

Infection of human erythroleukemic K562 cells by encephalomyocarditis virus readily resulted in establishment of persistently infected cultures. In contrast to the usual typical lytic infection by encephalomyocarditis virus, in which trypan blue staining of cells reaches close to 100% by about 15 h postinfection, K562 cell cultures required 3 to 4 days postinfection to reach a maximum of about 80 to 90% cell staining. The proportion of K562 cells taking up stain gradually decreased to about 10% of those present by about 13 days postinfection; during this time, virus yield per day measured by either plaque or hemagglutination titration fell about 10-fold. The decrease in percent staining was followed by waves of increased staining accompanied by increased virus production. Virus-producing cultures were maintained for over 3 months. Evolution of both virus and cells accompanied establishment of persistence in that plaque size changed from about 7 mm in diameter for the original virus to less than 1.5 mm by day 20 postinfection and most of the cells cloned from persistently infected cultures were resistant to superinfection with the original virus. Resistance was due, at least in part, to reduced virus attachment in that binding of 3H-labeled virus to cloned resistant cells was about 2% of that to uninfected cells.

Evidence for a direct role for sialic acid in the attachment of encephalomyocarditis virus to human erythrocytes.

Sialic acid residues are required in cellular receptors for many different mammalian viruses. Sialic acid could have a direct role, being an integral part of the virus binding site on the receptor. Alternatively, negatively charged sialic acid could have an indirect role, being responsible for holding the receptor in the required configuration for virus recognition, for instance, by interacting with positively charged amino acid residues found in the polypeptide chain of receptors. We have investigated the role of sialic acid in virus attachment by studying the interaction of the small RNA virus encephalomyocarditis (EMC) with glycophorin A, its receptor on human erythrocytes. In several experiments, influenza virus A was used for control purposes. Blocking positive charges on glycophorin either in lysine residues by acetylation or in arginine residues with butanedione did not affect its interaction with EMC virus. In contrast, blocking negatively charged carboxyl groups in sialic acid residues by amidation destroyed the ability of glycophorin to inhibit EMC virus attachment suggesting an important role for this part of sialic acid in EMC virus attachment. Removal of the polyhydroxy side chain in sialic acid residues of glycophorin by mild oxidation with periodate followed by reduction with borohydride had little effect on its interaction with EMC virus. Further, sialic acid species with either an acetyl or glycolyl group attached to the amino group on position 5 interacted equally well with EMC virus.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of production of multiple mRNAs for human glycophorin A.

The major sialoglycoprotein in the human red cell surface membrane, glycophorin A is encoded by a single gene. However, this gene gives rise to three species of glycophorin A mRNA of sizes about 1.0, 1.7 and 2.8 kilobases in reticulocytes, foetal liver cells and erythroleukaemic K562 cells. In an investigation of how the three mRNAs originated, we showed by primer extension analysis that all three mRNAs in K562 cells had identical 5' termini and, by nucleotide sequencing of correlated cDNAs, that they had identical coding regions, except for the well-known glycophorin AM-AN polymorphism. However, we found also by sequencing the cDNAs that the mRNAs apparently differed from each other in the lengths of their 3' untranslated regions. This was confirmed by Northern blot analysis which also provided evidence that the three mRNAs originated by use of different polyadenylation signals of which seven were found in the longest cDNA we analyzed.

Genetic stability of Ross River virus during epidemic spread in nonimmune humans.

We have examined the rate of evolution of Ross River virus, a mosquito-borne RNA virus, during epidemic spread through tens of thousands of nonimmune humans over a period of 10 months. Two regions of the Ross River virus genome were sequenced: the E2 gene (1.2 kb in length), which encodes the major neutralization determinant of the virus, and 0.4 kb of the 3'-untranslated region. In the E2 gene, a single nucleotide change was selected which led to a predicted amino acid change at residue 219. No changes were selected in the 3'-untranslated region. By comparison with rates of evolution reported for non-arthropod-borne RNA viruses, the rate for Ross River virus is surprisingly low. We identify three features of the Ross River virus replication and transmission cycle which may limit the rate of evolution of arthropod-borne viruses in the field.

Analysis of the bond between encephalomyocarditis virus and its human erythrocyte receptor by affinity chromatography on virus-sepharose columns.

Affinity chromatography was used to analyse the bond between encephalomyocarditis (EMC) virus and glycophorin, the receptor for EMC virus on human erythrocytes. Between 60 and 80% of glycophorin added to virus-Sepharose columns was retained compared with 10 to 20% retention on glycine-Sepharose columns. Elution with 0.2 M-NaCl released about 80 to 90% of the retained glycophorin from virus-Sepharose columns but little from glycine-Sepharose. Glycophorin remaining on either the virus or glycine columns after 0.2 M-NaCl treatment was released with Triton X-100, suggesting that this material was aggregated and trapped non-specifically. The sensitivity of the EMC virus-glycophorin bond to 0.2 M-NaCl was confirmed by showing that the radiolabelled EMC virus that bound to human erythrocyte membranes in 0.02 M-phosphate buffer was released when washed with this buffer containing 0.2 M-NaCl. It was concluded that weak ionic interactions were involved in this virus-receptor bond. Treatment of glycophorin with neuraminidase prevented it binding to EMC virus-Sepharose indicating the requirement for sialic acid for receptor activity. However, other sialylated molecules did not bind. Only one chymotryptic peptide of glycophorin (CH0) bound to EMC virus-Sepharose, confirming that this peptide contains the virus-binding site as had been previously suggested using other techniques. The effects of two non-ionic detergents and two anionic detergents on the virus-glycophorin bond were examined. In each case a very low concentration of detergent disrupted the bond and the concentration required was in parallel with that which dissolved erythrocyte membranes. It appears that multivalent glycophorin aggregates are required for stable bond formation.

Site of attachment of encephalomyocarditis virus on human erythrocytes.

Previous studies of the attachment of encephalomyocarditis (EMC) virus to human erythrocytes concluded that the glycophorins, a family of human erythrocyte sialoglycoproteins, act as EMC virus receptors. Evidence is presented that the major glycophorin species, glycophorin A, is the receptor for EMC virus attachment to human erythrocytes. Comparison of the structures of glycophorins A and B and sialoglycopeptides released by chymotrypsin and trypsin treatment of erythrocytes confirmed our previous suggestion (A. T. H. Burness and I. U. Pardoe, J. Gen. Virol. 64:1137-1148, 1983) that attachment of EMC virus to glycophorin A involves the region containing amino acids 35 to approximately 70 (numbered from the NH2 terminus), four of which (amino acids 37, 44, 47, and 50) are glycosylated. In addition, we provide evidence that the segment containing amino acids 35 to 39 with an oligosaccharide side chain on threonine-37 is particularly important for EMC virus attachment.

Aggregation of encephalomyocarditis virus induced by radio-iodination.

Radio-iodination causes encephalomyocarditis virus to behave aberrantly when examined by affinity chromatography and to sediment rapidly during analysis on sucrose density gradients suggesting that aggregation had taken place. The change in physical properties of the virus occurred whether iodination was carried out with 125I or 131I, with radio-iodine from two different sources, or using two different iodination procedures. The changes were not observed in virus subjected to an iodination procedure in the absence of radio-iodine suggesting that modification of tyrosine residues was involved rather than a side reaction such as amino acid oxidation. It is recommended that caution be exercised when following the fate of radio-iodinated virus in any particular study because its behaviour may not reflect that of normal, non-iodinated virus present.

A sialoglycopeptide from human erythrocytes with receptor-like properties for encephalomyocarditis and influenza viruses.

Encephalomyocarditis and influenza viruses attach to human erythrocytes causing haemagglutination. The receptor for both viruses on these cells is the major membrane sialoglycoprotein, glycophorin, solubilized preparations of which inhibit haemagglutination by either virus. We show here that glycophorin preparations inhibited haemagglutination of both viruses, even after the preparations were digested with chymotrypsin. To determine which component(s) in the digest exhibited activity, peptides separated by gel filtration were assayed for haemagglutination inhibition; one peptide only, CH-0, was active. A tentative structure was deduced for CH-0 from amino acid and sialic acid analyses. It was already known that neuraminidase treatment of erythrocytes or glycophorin prevents interaction with either virus, suggesting that sialic acid may form part of the active binding site in the receptor. However, receptor activity requires more than the presence of a particular arrangement of sialic acid since the arrangement in CH-0 was identical to that in two other inactive chymotryptic peptides. Examination by gel filtration, sucrose density gradient centrifugation and SDS-polyacrylamide gel electrophoresis demonstrated that Ch-0 readily aggregated, unlike the inactive peptides. It was proposed that the CH-0 chymotryptic peptide showed receptor-like activity (inhibited haemagglutination) because its tendency to aggregate allowed strong multivalent binding with virus particles.

Chromatofocusing of sialoglycoproteins.

Sialoglycoproteins of different sialic acid contents have been separated from each other by chromatofocusing on the ion exchanger PBE 94 using gradients of pH 4.00 down to pH 1.00. The technique is much faster than isoelectric focusing, apparently does not result in desialylation of the sialoglycoproteins and can handle with ease 10 ng to 50 mg quantities of protein on a 22 X 0.9 cm column. The technique revealed that commercial preparations of fetuin and human acid glycoprotein contained several components. Glycophorin, desialylated by controlled neuraminidase treatment, was fractionated by chromatofocusing into several components which differed in sialic acid content and in ability to inhibit haemagglutination by wheat germ agglutinin and encephalomyocarditis and influenza viruses.

The interaction of encephalomyocarditis virus with its erythrocyte receptor on affinity chromatography columns.

Glycophorin, the major sialoglycoprotein in the human erythrocyte surface membrane, can serve as a red cell receptor for both wheat-germ agglutinin (WGA) and encephalomyocarditis (EMC) virus since glycophorin bound to WGA--Sepharose can at the same time bind EMC virus. In contrast, glycophorin bound to WGA--Sepharose cannot bind EMC virus in the presence of SDS. The evidence suggests that virus binding to glycophorin-WGA--Sepharose occurred in the absence of SDS because glycophorin was present in aggregated complexes which were large enough either to accommodate both EMC virus and WGA at the same time, or alternatively to provide sufficient attachment sites for multivalent binding of virions.

Effect of enzymes on the attachment of influenza and encephalomyocarditis viruses to erythrocytes.

Encephalomyocarditis (EMC) and influenza viruses attach to human erythrocytes causing haemagglutination of the cells. Sialoglycoproteins, containing predominantly glycophorin A, from these cells behave as soluble virus receptors and inhibit haemagglutination by both viruses. Removal of 43% of the sialic acid from erythrocytes with neuraminidase prevented their haemagglutination by EMC virus loss of 40% of glycophorin sialic acid destroyed its inhibitory properties against this virus. However, about 80% of the sialic acid had to be removed from erythrocytes or from glycophorin to achieve the same results for influenza virus. Trypsin treatment of erythrocytes or glycophorin had little effect on haemagglutination or inhibition involving either virus, although the glycopeptides released contain up to 70% of the total sialic acid, and despite the fact that glycophorin was drastically reduced in size as shown by SDS--polyacrylamide gel electrophoresis. It is concluded that not all of the sialic acid present in erythrocyte sialoglycoprotein receptors is involved in attachment of EMC or influenza viruses and that the attachment sites on erythrocytes for these viruses are not identical.

Polyamines in encephalomyocarditis virus.

Encephalomyocarditis virus contains approximately 200 molecules of putrescine, 100 molecules of spermidine, and 40 molecules of spermine which could neutralize 11% of the viral genome. The same polyamines are present in different proportions in the Krebs ascites tumor cell in which the virus was grown.

Preparation of erythrocyte receptors for viruses by affinity chromatography.

A comparatively simple method for the purification of human erythrocyte receptors for encephalomyocarditis and influenza viruses is described. The procedure utilises the fact that these viruses share in common the erythrocyte receptor for wheat germ agglutinin (WGA), which enables commercially available WGA-Sepharose to be used in the purification of receptors for these viruses by affinity chromatography. Conditions are also described for introducing either 125I into the receptor in situ, or 3H-acetyl residues into the solubilised receptor.

The size and location of the poly(A) tract in EMC virus RNA.

Encephalomyocarditis (EMC) virus RNA, selected by its affinity for oligo(dT)-cellulose, contains poly(A) of size : (i) about 14 nucleotide residues long, based on the percentage of radioactivity in the RNA resistant to digestion by a mixture of pancreatic and T1 RNases; (ii) about 15 residues long, as measured by the ratio of the amount of terminal adenosine to internal adenylic acid in isolated poly(A); and (III) in the range 12 to 45 residues, the majority of tracts being about 16 to 18 residues long, based upon electrophoretic mobility on polyacrylamide gels using poly(A) molecules of known size as mol. wt. markers. The poly(A) appears to be located at the 3'-terminus of the virus genome since the tract, liberated by digestion with a mixture of pancreatic and T1 RNases, was shown by compositional analysis to contain a non-phosphorylated 3'-terminus and only adenine residues. The size heterogeneity in the poly(A) tracts revealed by gel electrophoresis is also consistent with a terminal location. Comparison of our data for EMC virus with published data for other picornaviruses suggests that the sizes of poly(A) tracts in polio- and Mengovirus RNA have been overestimated; poly(A) tracts in cardioviruses appear to be smaller than those in poliovirus; the minimum size of poly(A) required for full infectivity of picornavirus RNA has also been overestimated; a tract of at least 13 adenine residues long is required for full infectivity of EMC virus RNA.

Requirement of an adenylic acid-rich segment for the infectivity of encephalomyocarditis virus RNA.

About 80% of the RNA molecules extracted from encephalomyocarditis (EMC) virus were bound by oligo(dT)-cellulose under conditions which bind poly(A) but not poly(C) nor ribosomal RNA. This shows that most EMC virus RNA molecules contain a poly(A) tract. Both bound and unbound fractions contained RNA molecules of apparently the same size when examined by sucrose gradient sedimentation, but the bound fraction contained an adenylic acid-rich segment of about 20 nucleotides long, whereas the unbound RNA did not. The bound RNA had 200 times the specific infectivity of the unbound RNA which suggests that the poly(A) tract present in EMC virus RNA is required for infectivity.

Chromatographic studies on picornavirus capsid polypeptides.

The polypeptides of encephalomyocarditis, Mouse-Elberfeld and type 5 rhinoviruses behave similarly when chromatographed on calcium phosphate (brushite), each being eluted by a linear phosphate buffer gradient containing sodium dodecyl sulphate in three major peaks, CI, C2 and C3. Analysis of the peaks by polyacrylamide gel electrophoresis suggests that the major capsid polypeptides of these three picornaviruses elute in the order: delta (peak CI), gamma with (peak C2) and alpha (peak C3).