Detection of viral superantigen-class II MHC interactions at the cell surface.
Mouse mammary tumor virus superantigens (vSAgs) must bind to class II MHC proteins to activate T cells. Although direct interaction of vSAgs with class II proteins has been demonstrated biochemically, the details of this interaction are largely unresolved. To facilitate the study of class II-vSAg interactions, a sensitive assay has been developed that can detect binding of vSAgs to class II proteins on the cell surface. The assay measures changes in vSAg surface expression upon enzymatic removal of a co-expressed glycan-phosphatidyl inositol-anchored form of the class II molecule IE(k). Because the vSAgs are synthesized as integral membrane proteins that undergo proteolytic processing, an event that is likely required to eliminate membrane tethering, the data provide further evidence that a proteolytic fragment of vSAg is bound to class II proteins on the cell surface. The assay was utilized to identify mutant vSAgs that either did not associate with IE(k) molecules, or did not undergo furin-dependent proteolytic processing. Class II protein binding was detected using vSAg7 mutants that lacked furin endoprotease recognition sites, and after expression of vSAg in furin-deficient cells. The data demonstrate that furin-mediated processing is not necessary for association of vSAg7 with class II proteins, supporting previous studies that have indicated a role for alternative endoproteases in vSAg activation. However, because class II interactions were also noted in the apparent absence of proteases that are known to activate vSAgs, the data suggest that yet other proteases may process vSAgs in a fashion that does not necessarily lead to activation of T cells.
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Wadsworth Center, New York State Department of Health, PO Box 22002, 12201-2002, Albany, NY, USA.
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D000818:Animals; D017951:Antigen Presentation; D000938:Antigen-Presenting Cells / Q000276:immunology*; D000942:Antigens, Bacterial / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane; D006224:Cricetinae; D004768:Enterotoxins / Q000276:immunology; D045683:Furin; D017261:Glycosylphosphatidylinositols; D000949:Histocompatibility Antigens Class II / Q000276:immunology* / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D008324:Mammary Tumor Virus, Mouse / Q000276:immunology; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D013381:Subtilisins / Q000378:metabolism; D018089:Superantigens / Q000276:immunology* / Q000378:metabolism
D000942:Antigens, Bacterial; D000956:Antigens, Viral; D004768:Enterotoxins; D017261:Glycosylphosphatidylinositols; D000949:Histocompatibility Antigens Class II; D008562:Membrane Glycoproteins; C095378:Mtv-7 superantigen, Mouse mammary tumor virus; D018089:Superantigens; C026066:enterotoxin A, Staphylococcal; D013381:Subtilisins; D045683:Furin