THE CYTOTOXICITY OF THE PARVOVIRUS MINUTE VIRUS OF MICE NONSTRUCTURALPROTEIN NS1 IS RELATED TO CHANGES IN THE SYNTHESIS AND PHOSPHORYLATION OF CELL-PROTEINS

Autonomous parvoviruses exert lytic and cytostatic effects believed to contribute to their antineoplastic activity. Studies with inducible c lones have demonstrated a direct involvement of parvovirus nonstructur al proteins (NS) in oncolysis. Human and rat fibroblasts have been sta bly transfected with MVM(p) (minute virus of mice prototype strain) NS genes cloned under the control of a hormone-inducible promoter. Dexam ethasone-induced synthesis of the NS proteins in sensitive transformed cells results in cell killing within a few days. From these sensitive cell lines have been isolated some NS-resistant clones that also prov e resistant to MVM(p) infection, suggesting that cell factors modulate NS cytotoxicity. We have previously reported that factors involved in cell cycle regulation may contribute to this modulation, since NS tox icity requires cell proliferation and correlates with a cell cycle per turbation leading to an arrest in phase S/G(2). In addition to its rol e in cytotoxicity, NS1 can regulate transcription driven by parvovirus and nonparvovirus promoters. Since phosphorylation is a critical even t in controlling the activity of many proteins, notably transcription factors and cell cycle-regulated proteins, we have examined the effect of NS1 on the synthesis and phosphorylation of cell proteins. Our res ults indicate that NS1 interferes, within 7 h of induction, with phosp horylation of a protein of about 14 kDa (p14). Cell synchronization ha s enabled us to show that phosphorylation of this protein occurs in ea rly S phase and is prevented when NS1 is induced. This early effect of NS1 on p14 phosphorylation may be directly linked to cytotoxicity and is probably related to the previously reported inhibition of cell DNA synthesis. Late in the induction period (24 h), NS1 also alters the s ynthesis of a 50-kDa protein and a 35-kDa protein (p50 and p35, respec tively). Microsequencing of p35 reveals sequence homology with beta-tu bulin. These effects of NS1, observed only in NS1-sensitive cell lines , may be related to the protein's cytotoxicity.