title: Modulation of minute virus of mice cytotoxic activities through site-directed mutagenesis within the NS coding region.
abstract: Late in infection, parvovirus minute virus of mice (MVMp) induces the lysis of mouse A9 fibroblasts. This effect depends on the large nonstructural phosphoprotein NS1, which plays in addition a major role in viral DNA replication and progeny particle production. Since the NS1 C-terminal region is subjected to late phosphorylation events and protein kinase C (PKC) family members regulate NS1 replicative activities, the present study was conducted to determine the impact of PKCs on NS1 cytotoxic functions. To this end, we performed site-directed mutagenesis, substituting alanine residues for two consensus PKC-phosphorylation sites located within the NS1 C-terminal region, T585 and S588. Although these substitutions had no detectable effect on virus multiplication in a single-round infection, the NS1-585A mutant virus was significantly less toxic to A9 cells than wild-type MVMp, whereas the NS1-588A mutant virus was endowed with a higher killing potential. These alterations correlated with specific changes in the late phosphorylation pattern of the mutant NS1 proteins compared to the wild-type polypeptide. Since the mutations introduced in this region of the viral genome also made changes in the minor nonstructural protein NS2, a contribution of this polypeptide to the above-mentioned phenotypes of mutant viruses cannot be excluded at present. However, the involvement of NS1 in these phenotypes was directly supported by the respective reduced and enhanced capacity of NS1-585A and NS1-588A recombinant proteins for inducing morphological alterations and cell detachment in transfected A9 cultures. Altogether, these data suggest that late-occurring phosphorylation of NS1 specifically regulates the cytotoxic functions of the viral product and that residues T585 and S588 contribute to this control in an antagonistic way.
authors: Division F010, Applied Tumour Virology Program, and Institut National de la Santé et de la Recherche Médicale U375, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
authors: Laurent
authors: L
authors: Daeffler
authors: Rita
authors: R
authors: Hörlein
authors: Jean
authors: J
authors: Rommelaere
authors: Jürg P F
authors: JP
authors: Nüesch
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D051379:Mice; D008915:Minute Virus of Mice / Q000235:genetics / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D017931:DNA Primers; D017361:Viral Nonstructural Proteins