The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6

Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalyt ic kinase activity, and is present in infected cells as various isoforms ha ving molecular masses of 26, 28 and 30-34 kDa, We have previously shown tha t NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mu tants of the rotavirus YM NSP5 were constructed and assayed for their abili ty to interact with full-length NSP5 and NSP6 using the yeast two-hybrid as say. The homomultimerization domain was mapped to the 20 C-terminal aa of t he protein, which have a predicted alpha-helical structure. A deletion muta nt lacking the 10 C-terminal aa (Delta C10) failed to multimerize both in y east cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms), In cont rast, the Delta C10 mutant produced forms equivalent to the 26 and 28 kDa s pecies, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction do main with NSP6 was found to be present in the 35 C-terminal aa of NSP5, ove rlapping the multimerization domain of the protein, and suggesting that NSP 6 might have a regulatory role in the self-association of NSP5, NSP6 was al so found to interact with wild-type NSP5, but not with its mutant Delta C10 , in cells transiently transfected with plasmids encoding these proteins, c onfirming the relevance of the 10 C-terminal aa for the formation of the he terocomplex.