Dissecting the role of NSP5 in rotavirus replicative cycle by RNA interference

Rotavirus genomes contain 11 double stranded RNA segments. Genome segment 11 encodes for the non structural protein NSP5 and, in some strains, also for the non structural protein NSP6. NSP5 is produced soon after viral infection and undergoes a complex posttranslational hyperphosphorylation process that causes the generation of species characterised by reduced PAGE mobility. The use of deletion mutants demonstrated that NSP5 is able to autoregulating its own phosphorylation in a process that involves also the viral non structural protein NSP2 and cellular kinases. In particular, in vitro experiments had shown that casein kinase 1alpha (CK1 alpha) was the kinase responsible of phosphorylating NSP5 on serine 67 and that this was the first step in the process of hyperphosporylation. NSP5 localises in cytoplasmic viroplasms, structures where virus replication takes place. In viroplasms NSP5 has been shown to interact with the viral polymerase VP 1, the structural protein VP2 and the non structural one NSP2. In order to evaluate the role of NSP5 and its phosphorylation in the replicative cycle of rota virus, we used the RN A interference approach. In the first part of this thesis we targeted small interfering (si) RN As to genome segment 11 mRNA of two different rotavirus strains and were able to block NSP5 production in a strain-specific manner. This allowed us to demonstrate that lack of NSP5 had a strong effect on the overall viral replicative cycle causing inhibition of viroplasm formation, decreased production of other structural and non structural viral proteins, block of the synthesis of viral genomic dsRNA and production of infectious particles. In the second part of this thesis we wanted to evaluate if the results obtained on the hyperphosphorylation of NSP5 in vitro were confirmed in vivo and we decided to target a specific siRNA against CK1 alpha. This siRNA showed to be very efficient in blocking CK1 alpha production and allowed us to confirm in co-expression experiments that CK 1 alpha was the kinase responsible of phosphorylating NSP5 on serine 67. In addition we could show that in absence of CK1 alpha also the phosphorylation of NSP5 in the context of viral infection was impaired. Lack of NSP5 hyperphosphorylation did not affect the interaction ofNSP5 with the viroplasm resident proteins VPI and NSP2 and was not involved in virus protein production. However, had a strong effect on the viroplasms formation and morphogenesis causing the formation of viroplasms with altered shape and dimension. The results obtained demonstrate the essential role of NSP5 for the assembly of viroplasms, production of viral protein by de novo produced particle and virus replication. In addition the data presented confirm that also in vivo CK1 alpha is the kinase responsible of phosphorylating NSP5 and suggest that its hyperphosphorylation is important for the correct assembly of viroplasms