Journal
JOURNAL OF VIROLOGY
Volume 77, Issue 23, Pages 12466-12478Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.77.23.12466-12478.2003
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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-terminall 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-terminall 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 phosphoryllation 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 phosphoryllation 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.
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