PHOSPHORYLATION GENERATES DIFFERENT FORMS OF ROTAVIRUS NSP5

NSP5 (non-structural protein 5) is one of two proteins encoded by geno me segment 11 of group A rotaviruses. In virus-infected cells NSP5 acc umulates in the virosomes and is found as two polypeptides with molecu lar masses of 26 and 28 kDa (26K and 28K proteins), NSP5 has been prev iously shown to be post-translationally modified by the addition of O- linked monosaccharide residues of N-acetylglucosamine and also by phos phorylation, We have now found that, as a consequence of phosphorylati on, a complex modification process gives rise to previously unidentifi ed forms of NSP5, with molecular masses of up to 34 kDa, Treatment wit h phosphatases of NSP5 obtained from virus-infected cells produced a s ingle band of 26 kDa, NSP5 could be phosphorylated in vitro by incubat ion of immunoprecipitates with [gamma-P-32]ATP, producing mainly phosp horylated products of 28 and 32-34 kDa (32-34K), In both in vivo and i n vitro phosphorylated NSP5, phosphates were only found attached via s erine and threonine residues, The in vitro translated NSP5 precursor p olypeptide, molecular mass 25 kDa (25K), could also be phosphorylated and transformed into a 28K protein by incubation with extracts obtaine d from virus-infected cells, but not from non-infected cells, In addit ion, NSP5 labelled in vivo with [1,6-H-3]glucosamine showed mainly the presence of the 26K and 28K proteins (converted to 26K by protein pho sphatase treatment) suggesting that the type of protein produced is re gulated according to the level of phosphorylation and/or O-glycosylati on. The results also suggest that NSP5 is autophosphorylated.