Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms, where synthesis of the11 dsRNA genome segments and their packaging in new viral particles take place. RNA replication is mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2 and NSP2. We constructed a tagged form of VP1 and by coimmunoprecipitation experiments we demonstrated that VP1 and NSP5 interact in virus-infected cells as well as in the absence of other viral proteins or viral RNA in cotransfected cells. Using deletion mutants of NSP5 or different fragments of NSP5 fused to EGFP, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1. On the other hand, removal of the C-terminal 15 amino acids from tagged VP1 resulted in a less efficient coimmunoprecipitation with NSP5, suggesting an involvement of the Cterminus of VP1. Interaction of NSP5 with VP2 was investigated by coexpression of the two proteins in uninfected cells, which resulted in a strong hyperphosphorylation of NSP5 and in the formation of VLS, that we named VLS(VP2i) to distinguish them from those induced by NSP2, here designated as VLS(NSP2i). VLS(VP2i) were shown to assemble independently of the phosphorylation degree of NSP5 and to recruit the viroplasm-resident proteins NSP2, VP1, VP2 and VP6 (the protein forming the middle Abstract 4 layer of the virion). Attempts to coimmunoprecipitate NSP5 and VP2 failed both from infected and cotransfected cells. Tagged VP1 was found to localize in VLS (both VP2i and NSP2i) and in viroplasms, and to be able to replace wild-type VP1 structurally by being incorporated into progeny viral particles. Coexpression of different combinations of tagged VP1, NSP5, NSP2 and VP2 showed that the interaction of VP1 with NSP5 is not affected by the other viral proteins and is stronger than the interaction with NSP2. In addition, an inhibitory effect of VP1 on the levels of NSP5 hyperphosphorylation induced by both NSP2 and VP2 was observed. Altogether, these data confirmed an important role for NSP5 in replication, related with the interactions with the two structural proteins essentially involved in viral genome synthesis, and suggested that NSP5 plays a key role in architectural assembly of viroplasms and in recruitment of the other viroplasmic proteins.