Identification and characterization of a novel class of receptor for encephalomyocarditis virus on human cells

Jin, Yi-Ming (1993) Identification and characterization of a novel class of receptor for encephalomyocarditis virus on human cells. Masters thesis, Memorial University of Newfoundland.

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Abstract

Encephalomyocarditis (EMC) virus belongs to the genus cardiovirus of the picornavirus family. The virus is important in the etiological study of several virus-induced human diseases. However, little is known about EMC virus attachment to nucleated cells. In this thesis, the results on the characterization of the biochemical nature of EMC virus receptors on two human nucleated cell lines, K562 and HeLa cells, are presented. The study showed that EMC virus bound specifically to both cell types. The number of receptor sites on HeLa cells was in the range of 1.6 x 10⁵ per cell and the dissociation constant for virus binding was 1.1 nM. These results are consistent with previously determined values for the binding of EMC virus to K562 cells. After trypsin digestion of K562 and HeLa cells, regeneration of virus binding activity was inhibited by cycloheximide treatment, suggesting that recovery of the EMC virus-specific receptor is dependent upon intracellular protein synthesis. Further, digestion with proteases and neuraminidase, as well as lectin treatment of intact cells, cell membrane preparations and detergent-solubilized cell membranes, demonstrated that receptors for the virus on both K562 and HeLa cells are sialoglycoproteins. Affinity chromatography employing EMC virus columns isolated 70-kD receptor proteins from K562 and HeLa cells. Virus overlay protein assay revealed that EMC virus specifically recognized only the 70-kD proteins in both the purified receptor preparations and in detergent solubilized cell membranes, suggesting that virus attachment to K562 and HeLa cells could be exclusively mediated by the identified receptor molecules. Using the chromatofocusing technique, it was found that the receptor on K562 cells is likely more sialylated than that on HeLa cells. Finally, Western blot analysis using anti-glycophorin A antibody revealed that the antibody does not recognize the EMC virus 70-kD receptor on K562 or HeLa cells (the latter does not express glycophorin A). This indicates that the identified receptor proteins may not be glycophorin A, but they represent novel, not yet described EMC virus receptor molecules.

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