There are a number of discrepancies in the literature regarding the protein composition of the avian reoviruses. The present study demonstrates that avian reovirus S1133 contains at least 10 proteins (A, B, C, A, B, BC, BN, A, B, and C). Polypeptides B, BC, BN, B, and C are components of the outer capsid layer of the virus, while A, B, A, and A are core polypeptides. Protein C is a component of both layers, extending from the inner core to the outer capsid. The minor outer-capsid polypeptide C is shown to be the cell attachment protein, since it is the only viral polypeptide present in extracts of S1133infected cells that binds specifically to chicken embryo fibroblasts; furthermore, its binding to avian cells was competitively inhibited by S1133 reovirions but not by mammalian reovirions. Our results also show that C is an oligomeric protein both in the virion and free in the cytoplasm, and preliminary results suggest that the multimer is made up of three monomeric units. *Corresponding author. Phone and fax: 34-81-599157. 59 on July 10, 2020 by guest http://jvi.asm.org/ Downloaded from 60 MARTÍNEZ-COSTAS ET AL.

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.

Previous work has shown that the avian reovirus cell-attachment sigma C (σC) protein is a multimer. In the first part of this study the oligomerization state of intracellularly synthesized σC was analysed by different approaches, including SDS-PAGE, chemical cross-linking, sedimentation and gel filtration analysis. All these approaches indicated that protein σC in its native state is a homotrimer. In the second part of the present work we investigated the effect of different factors and reagents on oligomer stability, in order to elucidate the nature of the forces that maintain the conformational stability of the homotrimer. Our results, based on the stabilizing effect conferred by reducing agents, demonstrate that the σC subunits are not covalently bound via disulfide linkages. They further suggest that the formation of an intrachain disulfide bond between the two cysteine residues of the σC polypeptide has a negative effect on oligomer stability. The susceptibility of the trimer to pH, temperature, ionic strength, chemical denaturants and detergents indicates that hydrophobic interactions contribute much more to oligomer stability than do ionic interactions and hydrogen bonding. Finally, our results also reveal that mammalian and avian reovirus cell attachment proteins follow different subunit dissociation pathways.

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