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.
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Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada.
Pardoe
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Burness
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Michalak
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D015153:Blotting, Western; D002846:Chromatography, Affinity; D003513:Cycloheximide / Q000494:pharmacology; D004686:Encephalomyocarditis virus / Q000502:physiology*; D006021:Glycophorins / Q000032:analysis; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007526:Isoelectric Point; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis*; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D011991:Receptors, Virus / Q000032:analysis*; D012795:Sialoglycoproteins / Q000032:analysis*; D014407:Tumor Cells, Cultured
D006021:Glycophorins; D008562:Membrane Glycoproteins; D011991:Receptors, Virus; D012795:Sialoglycoproteins; D003513:Cycloheximide; D009439:Neuraminidase