Oligomerization and Cell-Binding Properties of the Avian Reovirus Cell-Attachment Protein ςC

Abstract

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.

The p17 Nonstructural Protein of Avian Reovirus Triggers Autophagy Enhancing Virus Replication via Activation of Phosphatase and Tensin Deleted on Chromosome 10 (PTEN) and AMP-activated Protein Kinase (AMPK), as well as dsRNA-dependent Protein Kinase (PKR)/eIF2α Signaling Pathways

“…It was proved that p17 plays an important role in regulating cell cycle and host cellular translation but does not induce cellular apoptosis (2)(3)(4). The C protein is encoded by the third ORF of the S1 segment, which is responsible for host cell attachment (5) and induction of apoptosis (2,6). Moreover, after initial binding of C protein to receptor(s) on the cell membrane, ARV enters their host cells using a caveolin-1-mediated and dynamin-2-dependent endocytic pathway that requires activation of p38 and Src signaling pathways (7).…”

Cell Entry of Avian Reovirus Follows a Caveolin-1-mediated and Dynamin-2-dependent Endocytic Pathway That Requires Activation of p38 Mitogen-activated Protein Kinase (MAPK) and Src Signaling Pathways as Well as Microtubules and Small GTPase Rab5 Protein

“…More recently, the nonstructural protein p17 is reported to regulate the cell cycle and host cellular translation (24). The avian reovirus fiber is formed by capsid protein C and is encoded by the third open reading frame of the S1 segment, which is responsible for host cell attachment (25,26) and induction of apoptosis (27).…”

Modification of Late Membrane Permeability in Avian Reovirus-infected Cells

“…1). All three S1 ORFs are expressed in infected cells (11,12); ORFs 1 and 2 direct the synthesis of the nonstructural proteins p10 and p17, which associate with cell membranes, whereas distal ORF3 expresses protein C, a minor trimeric structural protein that is involved in virus attachment to cell receptors (13,14). Recent evidence indicates that there is a close association between nonstructural p10 protein and the syncytial phenotype displayed by avian reoviruses (11,15).…”