Mutagenesis within the NS Coding Region Cytotoxic Activities through Site-Directed Modulation of Minute Virus of Mice

Late in infection, parvovirus minute virus of mice (MVMp) induces the lysis of mouse A9 fibroblasts. Thiseffect depends on the large nonstructural phosphoprotein NS1, which plays in addition a major role in viralDNA replication and progeny particle production. Since the NS1 C-terminal region is subjected to latephosphorylation events and protein kinase C (PKC) family members regulate NS1 replicative activities, thepresent study was conducted to determine the impact of PKCs on NS1 cytotoxic functions. To this end, weperformed site-directed mutagenesis, substituting alanine residues for two consensus PKC-phosphorylationsites located within the NS1 C-terminal region, T585 and S588. Although these substitutions had no detectableeffect on virus multiplication in a single-round infection, the NS1-585A mutant virus was significantly less toxicto A9 cells than wild-type MVMp, whereas the NS1-588A mutant virus was endowed with a higher killingpotential. These alterations correlated with specific changes in the late phosphorylation pattern of the mutantNS1 proteins compared to the wild-type polypeptide. Since the mutations introduced in this region of the viralgenome also made changes in the minor nonstructural protein NS2, a contribution of this polypeptide to theabove-mentioned phenotypes of mutant viruses cannot be excluded at present. However, the involvement ofNS1 in these phenotypes was directly supported by the respective reduced and enhanced capacity of NS1-585Aand NS1-588A recombinant proteins for inducing morphological alterations and cell detachment in transfectedA9 cultures. Altogether, these data suggest that late-occurring phosphorylation of NS1 specifically regulatesthe cytotoxic functions of the viral product and that residues T585 and S588 contribute to this control in anantagonistic way.