Importance of African horse sickness virus NS2 protein phosphorylation for the formation of viral inclusion bodies

Abstract

African horse sickness virus (AHSV), a member of the Orbivirus genus (family Sedoreoviridae, order Reovirales), encodes seven structural proteins and four non-structural proteins. The non-structural protein NS2 is the only virus-specified phosphoprotein and is the main component of the characteristic virus inclusion bodies (VIBs) observed in the cytoplasm of orbivirus-infected cells. Although VIBs are considered the sites at which virus replication and assembly occurs, the factors that govern their formation is still poorly understood in the case of AHSV. By making use of in vitro phosphorylation assays, kinase inhibitor studies, phosphoprotein staining assays and immunofluorescent confocal microscopy, wild-type NS2 and NS2 mutant proteins were characterized. Based on the results, it can be concluded that NS2 of AHSV-4 is phosphorylated by Casein kinase 2 at serine residues 256 and 258, and that phosphorylation of the NS2 protein is required for the formation of intracellular virus inclusion body-like structures.