IN-VIVO AND IN-VITRO PHOSPHORYLATION OF ROTAVIRUS NSP5 CORRELATES WITH ITS LOCALIZATION IN VIROPLASMS

NSP5 (NS26), the product of rotavirus gene 11, is a phosphoprotein who se role in the virus replication cycle is unknown. To gain further ins ight into its function, we obtained monoclonal antibodies against the baculovirus-expressed protein. By immunoprecipitation and immunoblotti ng experiments, we showed that (i) NSP5 appears in many different phos phorylated forms in rotavirus-infected cells; (ii) immunoprecipitated NSP5 from rotavirus-infected cells can be phosphorylated in vitro by i ncubation with ATP; (iii) NSP5, produced either by transient transfect ion of rotavirus gene 11 or by infection by gene 11 recombinant vaccin ia virus or baculovirus, can be phosphorylated in vivo and in vitro; ( iv) NSP5 expressed in Escherichia coli is phosphorylated in vitro, and thus NSP5 is a potential protein kinase; and (v) NSP5 forms dimers an d interacts with NSP2. The intracellular localization of NSP5 in the c ourse of rotavirus infection and after transient expression in COS7 ce lls has also been investigated. In rotavirus-infected cells, NSP5 is l ocalized in viroplasms, but it is widespread throughout the cytoplasm of transfected COS7 cells. NSP5 produced by transfected COS7 cells did not acquire the multiphosphorylated forms observed in rotavirus-infec ted COS7 cells. Thus, there is a tight correlation between the localiz ation of NSP5 in the viroplasms and its protein kinase activity in viv o oi in vitro. Our results suggest that cellular or viral cofactors ar e indispensable to fully phosphorylate NSP5 and to reach its intracell ular localization.