Towards determining the functional importance of African horse sickness virus NS2 protein phosphorylation

Abstract

The aim of this investigation was to obtain more information regarding the phosphorylation of the non-structural protein NS2 of African horse sickness virus (AHSV), a member of the Orbivirus genus within the Reoviridae family. The NS2 protein is the only virus-specified phosphoprotein, it is capable of binding single-stranded RNA and it is also the main component of the characteristic virus inclusion bodies observed in the cytoplasm of orbivirusinfected cells. These properties of NS2 suggest that it may play an important role in viral replication and morphogenesis. It has been reported that the NS2 protein of different orbiviruses is phosphorylated on serine residues within its carboxy (C) terminus, most likely by the ubiquitous cellular kinase casein kinase II (CK2). Bioinformatic analysis of the AHSV-9 NS2 amino acid sequence led to the identification of three C-terminal serine residues at positions 256, 258 and 262 that were predicted to be CK2 phosphorylation sites. The importance of these serine residues for phosphorylation of the AHSV-9 NS2 protein was subsequently investigated by analysis of recombinant baculovirus-expressed mutant versions of the NS2 protein. These comprised of single and dual mutant NS2 proteins in which the identified serine residues were respectively substituted either with alanine or with negatively charged aspartic acid residues to mimic phosphoserine residues. Since it has been reported that phosphorylation of the NS2 protein of bluetongue virus, the prototype orbivirus, influences its ability to form virus inclusion bodies (VIBs), it was investigated whether differences could be observed in the inclusion bodies formed by the respective mutant AHSV-9 NS2 proteins. Transmission electron microscope analysis of recombinant baculovirus-infected Spodoptera frugiperda insect cells indicated that a mutant NS2 protein in which the serine residues at both positions 256 and 258 were substituted with alanine was unable to form cytoplasmic inclusions. However, when both of these residues were substituted with aspartic acid residues, inclusion bodies could be observed and thus suggested that both of these serine residues may serve as phosphate-acceptor sites. To further investigate whether NS2 protein phosphorylation may be important for the formation of VIBs, Culicoides variipennis insect cells were treated with myricetin, an inhibitor of CK2 activity, prior to infection with AHSV-9. In contrast to the untreated virus-infected cells, immunofluorescent confocal laser microscopy using a monospecific anti-NS2 antibody revealed no distinctive NS2 aggregates in the treated cells. Furthermore, immunofluorescence analysis of bromouridine (BrU)-labelled RNA made in infected cells indicated that it localizes to the VIBs. Overall, the results obtained during the course of this investigation suggest that NS2 phosphorylation of serine residues is mediated by CK2 and that this posttranslational modification of the NS2 protein is required for the formation of VIBs in AHSVinfected cells.