A Genetically Engineered Rotavirus NSP2 Phosphorylation Mutant Impaired in Viroplasm Formation and Replication Shows an Early Interaction between vNSP2 and Cellular Lipid Droplets

Abstract

Many RNA viruses replicate in cytoplasmic compartments (virus factories or viroplasms) composed of viral and cellular proteins, but the mechanisms required for their formation remain largely unknown. Rotavirus (RV) replication in viroplasms requires interactions between virus nonstructural proteins NSP2 and NSP5 that are associated with components of lipid droplets (LDs). We previously identified two forms of NSP2 in RV-infected cells; a cytoplasmically dispersed (dNSP2) and a viroplasm-specific (vNSP2) that interact with hypo- and hyper-phosphorylated NSP5, respectively, that indicates a coordinated phosphorylation cascade controls viroplasm assembly. The cellular kinase CK1α phosphorylates NSP2 on Serine 313 triggering the localization of vNSP2 to sites of viroplasm assembly and its association with hyper-phosphorylated NSP5. Using reverse genetics, we generated a rotavirus with a phosphomimetic NSP2 (S313D) mutation to directly evaluate the role of CK1α NSP2 phosphorylation on viroplasm formation. Recombinant rotavirus NSP2 S313D (rRV NSP2 S313D) is significantly delayed in viroplasm formation, virus replication, and interferes with wild type RV replication in co-infection. Taking advantage of the delay in viroplasm formation, the NSP2 phosphomimetic mutant was used as a tool to observe very early events in viroplasm assembly. We show that (1) viroplasm assembly correlates with NSP5 hyper-phosphorylation, and (2) that vNSP2 S313D co-localizes with RV-induced LDs without NSP5, suggesting that vNSP2 phospho-S313 is sufficient for interacting with LDs and may be the virus factor required for RV-induced LD formation. Further studies with the rRV NSP2 S313D virus are expected to reveal new aspects of viroplasm and LD initiation and assembly. IMPORTANCE Reverse genetics was used to generate a recombinant rotavirus with a single phosphomimetic mutation in nonstructural protein 2 (NSP2 S313D) that exhibits delayed viroplasm formation, delayed replication, and an interfering phenotype during co-infection with wild type rotavirus, indicating the importance of this amino acid during virus replication. Exploiting the delay in viroplasm assembly, we found that viroplasm-associated NSP2 co-localizes with rotavirus-induced lipid droplets prior to the accumulation of other rotavirus proteins that are required for viroplasm formation, and that NSP5 hyper-phosphorylation is required for viroplasm assembly. These data suggest that NSP2 phospho-S313 is sufficient for interaction with lipid droplets and may be the virus factor that induces lipid droplet biogenesis in rotavirus-infected cells. Lipid droplets are cellular organelles critical for the replication of many viral and bacterial pathogens, and thus, understanding the mechanism of NSP2-mediated viroplasm/lipid droplet initiation and interaction will lead to new insights into this important host-pathogen interaction.