title: Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2.
abstract: We have previously shown that a number of isoforms of the non-structural rotavirus protein NSP5 are found in virus-infected cells. These isoforms differ in their level of phosphorylation which, at least in part, appears to occur through autophosphorylation. NSP5 co-localizes with another non-structural protein, NSP2, in the viroplasms of infected cells where virus replication takes place. We now show that NSP5 can be chemically cross-linked in living cells with the viral polymerase VP1 and NSP2. Interaction of NSP5 with NSP2 was also demonstrated by co-immunoprecipitation of NSP2 and NSP5 from extracts of UV-treated rotavirus-infected cells. In addition, in transient transfection assays, NSP5 phosphorylation in vivo was enhanced by co-expression of NSP2. An NSP5 C-terminal domain deletion mutant, was completely unable to be phosphorylated either in the presence or absence of NSP2. However, a 33 aa N-terminal deletion mutant of NSP5 was shown to become hyperphosphorylated in vivo and to be insensitive to NSP2 activation, suggesting a regulatory role for this domain in NSP5 phosphorylation and making it a candidate for the interaction with NSP2. These mutants also allow a preliminary mapping of NSP5 autophosphorylation activity.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Padriciano, Trieste, Italy.
authors.forename: I
authors.initials: I
authors.lastname: Afrikanova
authors.forename: E
authors.initials: E
authors.lastname: Fabbretti
authors.forename: M C
authors.initials: MC
authors.lastname: Miozzo
authors.forename: O R
authors.initials: OR
authors.lastname: Burrone
mesh_terms: D000818:Animals; D015967:Gene Expression Regulation, Viral; D009154:Mutation; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012401:Rotavirus / Q000378:metabolism*; D015854:Up-Regulation; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C079234:NS35 protein, rotavirus; C071649:gene 11 protein, Rotavirus B