Oligomerization and cell-binding properties of the avian reovirus cell-attachment protein sigmaC.
Avian reovirus protein sigmaC, the viral cell-attachment protein, is a minor component of the outer-capsid shell of the viral particle that is synthesized in small amounts in infected cells. We cloned the sigmaC-encoding ORF in vector pIL-2f, expressed it in Escherichia coli, and partially purified the resulting recombinant protein from inclusion bodies. Rabbit polyclonal antibodies raised against the recombinant protein specifically recognized the viral polypeptide in ELISA, immunoprecipitation, and Western blotting. To study the oligomerization capacity and cell-binding affinity of protein sigmaC, the sigmaC-encoding ORF was also expressed in chicken embryo fibroblasts (CEFs) and in reticulocyte lysates. In all three systems protein sigmaC is expressed as a multimer with identical electrophoretic mobility to the naturally occurring protein. Cell-binding experiments show that both in vitro and in vivo expressed protein sigmaC display affinity for CEF receptors, and this property is exclusively associated with the oligomeric form of the protein. The fact that incubation of CEF cells with the recombinant protein expressed in bacterial cells completely blocks the binding of purified reovirions indicates both that binding of this protein to cells is specific and saturable, and that reovirions and protein sigmaC bind to the same class of cell receptor. Saturation binding experiments, performed with the recombinant protein expressed in E. coli and with purified reovirions, showed that the number of cellular receptor sites (CRSs) for avian reovirus S1133 is 1.8 x 10(4) per CEF cell, whereas the number of cellular receptor units (CRUs) for sigmaC is 2.2 x 10(5) per CEF cell. These results are consistent with previous reports on the binding of mammalian reoviruses.
Grande
A
A
Departamento de Bioquímica y Biología Molecular, Universidad de Santiago de Compostela, Santiago de Compostela, 15706, Spain.
Rodriguez
E
E
Costas
C
C
Everitt
E
E
Benavente
J
J
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D004926:Escherichia coli / Q000235:genetics; D007106:Immune Sera / Q000276:immunology; D007700:Kinetics; D016366:Open Reading Frames / Q000235:genetics; D018165:Orthoreovirus / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011991:Receptors, Virus / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D007106:Immune Sera; D011991:Receptors, Virus; D011994:Recombinant Proteins; D014764:Viral Proteins