Regulation of MVM NS1 by protein kinase C: Impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the o nly viral nonstructural protein required in all cell types and it is involv ed in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosp horylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it ha s been demonstrated that NS1 replicative functions, in particular, DNA-unwi nding activities, are regulated by protein kinase C (PKC), as exemplified b y the modulation of NS1 helicase activity by PKC lambda phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, inc luding the induction of cytopathic effects, a mutational approach was pursu ed in order to produce NS1 variants harboring amino acid substitutions at c andidate PKC target residues. Besides the determination of two additional i n vivo phosphorylation sites in NS1, this mutagenesis allowed the segregati on of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being imp aired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduc ed cytotoxicity, leading to sustained gene expression, while keeping functi ons necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the c ourse of a viral infection, NS1 may undergo a shift from productive to cyto toxic functions as a result of a phosphorylation-dependent regulation. (C) 2000 Academic Press.