Two non-structural rotavirus proteins, NSP2 and NSP5, form viroplasm-like structures in vivo

In rotavirus-infected cells, the non-structural proteins NSP5 and NSP2 loca lize in complexes called viroplasms, where replication and assembly occur. Recently, we have demonstrated direct interaction of NSP5 with NSP2, and as a consequence of that, up-regulation of NSP5 hyperphosphorylation. To inve stigate a possible structural role for the NSP2-NSP5 interaction, we analys ed the cytoplasmic distribution of the two proteins in transfected cells by immunofluorescence using specific antibodies. Here we report that NSP2 and NSP5 can drive the formation of viroplasm-like structures (VLS) in the abs ence of other rotaviral proteins and rotavirus replication. Several NSP5 de letion mutants were constructed and expressed in combination with NSP2. Bot h the N- and C-terminal domains of NSP5 were found to be essential for VLS formation. Only one mutant, with an internal deletion of residues 81-130, w as able to interact with NSP2 to form VLS. Analysis of the phosphorylation capacity of the different mutants in vivo indicated that hyperphosphorylati on of NSP5 is necessary, but not sufficient, for VLS formation, Our results suggest a role for the non-structural protein NSP5 in the structure of vir oplasms mediated by its interaction with NSP2.