Cross-linking of a polyomavirus attachment protein to its mouse kidney cell receptor.
We used photoaffinity cross-linking with the heterobifunctional cross-linker N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) to covalently link polyomavirus to a mouse kidney cell surface component. The virus-HSAB combination was adsorbed to the cells and then cross-linked and isolated in monopinocytotic vesicles from the cells after endocytosis. The cross-linked product was identified on sodium dodecyl sulfate-polyacrylamide gels by the presence of a new band carrying 125I-labeled virion protein with a higher molecular mass than the normal virion protein bands. A single new band, with an apparent molecular mass of 120 kilodaltons (120 kDa), was identified by this procedure. This band was formed only in the presence of the HSAB cross-linker when virions were bound to the cells. The band also copurified with cross-linked virions when virion-containing vesicles were treated with detergent to remove the cell membrane. Antibody treatments that blocked up to 100% of virus binding and internalization also blocked cross-linking, as measured by the formation of the 120-kDa band. The 120-kDa band was characterized by preparation of antibody against the excised band from the gel. This antibody was shown to have the expected dual specificity for polyomavirus VP1 sequences and plasma membrane proteins, as analyzed on Western blots. The anti-120-kDa antibody was also shown by immunofluorescence to bind to the surface of mouse kidney cells. These data have demonstrated that molecules of possible biological significance in the binding of polyomavirus to mouse kidney cells have been cross-linked and that cell surface molecules have been identified that may be characterized further for possible receptor function in polyomavirus attachment.
D000268:Adhesiveness; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001345:Autoradiography; D001386:Azides / Q000494:pharmacology; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D007074:Immunoglobulin G / Q000276:immunology; D007457:Iodine Radioisotopes; D007668:Kidney / Q000382:microbiology; D051379:Mice; D008970:Molecular Weight; D011120:Polyomavirus / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D011991:Receptors, Virus / Q000378:metabolism*; D014764:Viral Proteins / Q000276:immunology / Q000378:metabolism*; D015678:Viral Structural Proteins
D000911:Antibodies, Monoclonal; D001386:Azides; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D011991:Receptors, Virus; D014764:Viral Proteins; D015678:Viral Structural Proteins; C029534:hydroxysuccinimidyl-4-azidobenzoate