doi:10.1128/JVI.79.1.184-192.2005. viroplasm formation. Recombinant rotavirus NSP2 S313D (rRV NSP2 S313D) is significantly delayed in viroplasm formation and in virus replication and interferes with wild-type RV replication in coinfection. 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 (i) viroplasm assembly correlates with NSP5 hyperphosphorylation and (ii) vNSP2 S313D colocalizes 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. Rabbit Polyclonal to DAPK3 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 Atopaxar hydrobromide 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 coinfection 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 Atopaxar hydrobromide colocalizes with rotavirus-induced lipid droplets prior to the accumulation of other rotavirus proteins that are required for viroplasm formation and that NSP5 hyperphosphorylation is required for viroplasm assembly. These data suggest that NSP2 phospho-S313 is sufficient for connection with lipid droplets and may be the disease element 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 connection will lead to fresh insights into this important host-pathogen connection. NSP2 autophosphorylation ability for NSP2 resulting in multiple phosphorylations throughout the NSP2 protein (17). Additionally, we showed that the cellular kinase CK1 phosphorylates NSP2 on serine 313 (S313) located in the C terminus -helical tail (C-terminal helix [CTH]; amino acids [aa] 295 to 317) (17). Phosphorylation of S313 appears preferential on vNSP2, becoming phosphorylated at a rate 40 times more than that seen with dNSP2 (9). The CTH of NSP2 is required for viroplasm formation (9) and was expected to facilitate the formation of interoctamer NSP2 chains through website swapping, or connection, with the CTH on neighboring NSP2 octamers (13). Our crystallographic analysis of a recombinant phosphomimetic NSP2 protein (NSP2 S313D) showed that phosphorylation of NSP2 S313 likely catalyzes four interoctamer hydrogen bridges between adjacent CTHs of neighboring octamers (aspartic acid 313octamer A-arginine 287octamer B), providing strong evidence that CK1 phosphorylation of NSP2 could be the catalyst for viroplasm assembly (17). NSP5 is definitely a RV (17). On the basis of this crystallography result, we hypothesized that a RV NSP2 S313D phosphomimetic mutant disease would act as a gain-of-function mutant and be able to catalyze the faster assembly of viroplasms and thus enhance disease replication. To test this hypothesis, we generated two recombinant RVs (rRV): (i) a rRV having a wild-type genome (rRV WT) and (ii) a recombinant RV with a single genome mutation in gene section 8 encoding NSP2 to generate a phosphomimetic NSP2 S313D disease (rRV NSP2 S313D) Atopaxar hydrobromide (19). The rRV WT disease was acquired using the standard 7-day passage explained in the protocol published by Kanai et al. (19) Recovery of rRV NSP2 S313D, expected to be a gain-of-function mutant, was accomplished only after an extended incubation of the second passage over a period of 5 weeks of uninterrupted incubation time. Both rRV WT and rRV NSP2 S313D viruses were then amplified to high titer. The phosphomimetic rRV NSP2 S313D disease is viable but shows delayed replication kinetics and a small-plaque phenotype compared to rRV WT disease. The prolonged incubation time needed to acquire the rRV NSP2 S313D mutant disease suggested that this disease was not a gain-of-function mutant. To determine the fitness of the rRV NSP2 S313D disease compared to the rRV.