Synthesis of Bluetongue Virus-encoded Phosphoprotein and Formation of Inclusion Bodies by Recombinant Baculovirus in Insect Cells: It Binds the Single-stranded RNA Species

A DNA clone of RNA segment 8 (S8) of bluetongue virus type 10 (BTV-10), an orbivirus member of the Reoviridae family has been expressed to high levels (20 mg/1 x 10(9) cells) using an Autographa californica nuclear polyhedrosis virus expression vector (pA-cYM1). The expressed protein is similar to the authentic BTV phosphoprotein NS2, in its size, antigenicity, and also the manner of phosphorylation (e.g. same peptides and residues). Both mammalian and insect cell-derived NS2 proteins are phosphorylated at serine residues only. Using affinity column chromatography and a gel retardation assay, the expressed protein has been shown to possess ssRNA-binding ability, a property which is shown to be independent of the phosphorylation state of the protein. In immunoelectron micrographic studies, gold-labelled anti-expressed NS2 antibodies have been used to localize the NS2 protein within the viral inclusion bodies (VIBs) in BTV-infected mammalian cells. Large inclusion bodies, morphologically similar to VIBs, have been identified in the recombinant virus-infected Spodoptera frugiperda cells. These structures have been shown to react with gold-labelled anti-BTV-10 antisera, demonstrating the first direct evidence of the origin of inclusion bodies in orbivirus infection.