A Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus Replication

Many viruses use specific viral proteins to bind calcium ions (Ca(2+)) for stability or to modify host cell pathways; however, to date, no Ca(2+) binding protein has been reported in bluetongue virus (BTV), the causative agent of bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca(2+) binding motif in the carboxyl terminal region of BTV nonstructural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca(2+) efficiently and that Ca(2+) binding was perturbed when the Asp and Glu residues in the motif were substituted by alanine. Using circular dichroism analysis, we found that Ca(2+) binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in the presence of Ca(2+) revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain that wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca(2+) enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca(2+) binding site in the viral genome by reverse genetics failed to allow recovery of viable virus, and the NS2 phosphorylation level and assembly of viral inclusion bodies (VIBs) were reduced. Together, our data suggest that NS2 is a dedicated Ca(2+) binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly. IMPORTANCE After entering the host cells, viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells, members of the Reoviridae family form inclusion body-like structures known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium-binding EF-hand-like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.