Structure Function Studies of Rotavirus NSP5

Rotaviruses, causative agents of gastroenteritis in young animals and humans, are large icosahedral viruses with a complex architecture. The double-stranded RNA (dsRNA) genome composed of 11 segments, that codes for 6 structural and 6 non-structural proteins, is enclosed within three concentric capsid layers. NSP5, a non structural protein, is encoded by segment 11. It is produced early in infection and localizes in ‘viroplasms’, cytoplasmic inclusion bodies in which viral RNA replication and packaging take place. NSP5 is essential for the replicative cycle of the virus since, in its absence, viroplasms are not formed and viral RNA replication and transcription do not occur. NSP5 is known to undergo two different types of posttranslational modifications, a cytoplasmic O-glycosylation and phosphorylation, which lead to the formation of proteins differing in electrophoretic mobility. Although the hyperphosphorylation process of NSP5 seems to be very complex, its role in the replicative cycle of rotavirus is unknown. We demonstrated that NSP5 operates as an auto-regulator of its own phosphorylation as a consequence of two distinct activities of the protein: substrate and activator. In the first part of the thesis we have shown, that phosphorylation of Ser-67 within the SDSAS motif (amino acids 63-67) was required to trigger hyperphosphorylation by promoting the activation function. The evidence coming from iv vitro experiments, including kinase assay with recombinant casein kinase 1α from zebrafish, proved that this enzyme is responsible for a key phosphorylation step that initiates the whole hyperphosphorylation cascade of NSP5. In the second part of the dissertation, using MALDI TOF/TOF spectroscopy, we added new data to the information about the posttranslational modifications of NSP5. We confirmed that the region of the protein encompassing Ser-67 is phosphorylated in vivo. Additionally we managed to map which parts of NSP5 sequence carries N-acetyloglucosamine and which regions bear phosphorylated serines or threonines. There is no evidence about structure of NSP5 so far. In the last chapter we focused on investigating the structural organization of this crucial viral protein. To achieve this, in addition to the full length protein, one point mutation and two different truncation mutants were constructed, expressed, purified and refolded. The secondary structure of the different proteins was analyzed by circular dichroism spectroscopy and general information about protein conformation was provided. Our findings, together with an analysis of NSP5 sequence indicate that NSP5 can be an intrinsically unfolded/disordered protein.