Plasmid-based Reverse Genetics for Probing Phosphorylation-dependent Viroplasm Formation in Rotaviruses

Rotavirus (RV) replication occurs in cytoplasmic compartments, known as viroplasms, that are composed of viral and cellular proteins. Viroplasm formation requires RV nonstructural proteins NSP2 and NSP5 and cellular lipid droplets (LDs); however, the mechanisms required for viroplasm assembly remain largely unknown. We previously identified two conformationally-distinct forms of NSP2 (dNSP2, vNSP2) found in RV-infected cells that interact differentially with hypo- and hyperphosphorylated NSP5, respectively, and indicate a coordinated phosphorylation-dependent mechanism regulating viroplasm assembly. We also reported that phosphorylation of dNSP2 on serine 313 by the cellular kinase CK1α triggers the localization of vNSP2 to sites of viroplasm assembly and its association with hyperphosphorylated NSP5. To directly evaluate the role of CK1α-mediated NSP2 phosphorylation on viroplasm formation, we used a recently published plasmid-based reverse genetics method to generate a recombinant rotavirus (rRV) with a phosphomimetic NSP2 mutation (rRV NSP2 S313D). The rRV NSP2 S313D virus is significantly delayed in viroplasm formation, virus replication, and interferes with wild type RV replication during co-infection. The rRV NSP2 S313A virus was not rescued. Taking advantage of the delay in viroplasm formation, the NSP2 S313D phosphomimetic mutant was used as a tool to observe very early events in viroplasm assembly. We show that (1) viroplasm assembly correlates with NSP5 hyperphosphorylation, 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.