期刊
VIROLOGY
卷 274, 期 2, 页码 367-377出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/viro.2000.0473
关键词
avian reovirus; protein sigma C; oligomerization; cell binding
类别
Avian reovirus protein sigma C, 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 sigma C-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 sigma C, the sigma C-encoding ORF was also expressed in chicken embryo fibroblasts (CEFs) and in reticulocyte lysates. in all three systems protein sigma C 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 sigma C 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 sigma C 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 sigma C is 2.2 x 10(5) per CEF cell. These results are consistent with previous reports on the binding of mammalian reoviruses. (C) 2000 Academic Press.
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