Oligomerization and cell-binding properties of the avian reovirus cell-attachment protein sigma C

Avian reovirus protein sigma C, the viral cell-attachment protein, is a min or component of the outer-capsid shell of the viral particle that is synthe sized in small amounts in infected cells. We cloned the sigma C-encoding OR F in vector pIL-2f, expressed it in Escherichia coli, and partially purifie d the resulting recombinant protein from inclusion bodies. Rabbit polyclona l 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 sigma C, the sigma C-encoding ORF was also expressed in chicken embryo fib roblasts (CEFs) and in reticulocyte lysates. in all three systems protein s igma C is expressed as a multimer with identical electrophoretic mobility t o the naturally occurring protein. Cell-binding experiments show that both in vitro and in vivo expressed protein sigma C display affinity for CEF rec eptors, and this property is exclusively associated with the oligomeric for m of the protein. The fact that incubation of CEF cells with the recombinan t protein expressed in bacterial cells completely blocks the binding of pur ified reovirions indicates both that binding of this protein to cells is sp ecific and saturable, and that reovirions and protein sigma C bind to the s ame class of cell receptor. Saturation binding experiments, performed with the recombinant protein expressed in E. coil 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 sigma C is 2.2 x 10(5) per CEF cell. These results are co nsistent with previous reports on the binding of mammalian reoviruses. (C) 2000 Academic Press.