REPLICATIVE FUNCTIONS OF MINUTE VIRUS OF MICE NS1 PROTEIN ARE REGULATED IN-VITRO BY PHOSPHORYLATION THROUGH PROTEIN-KINASE-C

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytoto xicity, transcriptional regulation, and initiation of viral DNA replic ation. For coordination of these various functions during virus propag ation, NS1 has been proposed to be regulated by posttranslational modi fications, in particular phosphorylation, Recent in vitro studies (J, P. F. Nuesch, R. Corbau, P. Tattersall, and J. Rommelaere, J, Virol, 7 2:8002-8012, 1998) provided evidence that distinct NS1 activities, not ably the intrinsic helicase function, are modulated by the phosphoryla tion state of the protein. In order to study the dependence of the ini tiation of viral DNA replication bn NS1 phosphorylation and to identif y the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on p lasmid substrates containing the minima! left-end origins of replicati on. Cellular components necessary to drive NS1-dependent rolling-circl e replication (RCR) were freed from endogenous serine/threonine protei n kinases by affinity chromatography, and the eukaryotic DNA polymeras es were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1(P)) supported RCR under these conditions, dephosphorylated NS 1 (NS1(O)) was impaired. Using fractionated HeLa cell extracts, we ide ntified two essential protein components which are able to phosphoryla te NS1(O), are enriched in protein kinase C (PKC), and, when present t ogether, reactivate NS1(O) for replication. One of these components, c ontaining atypical PKC, was sufficient to restore NS1(O) helicase acti vity. The requirement of NS1(O) reactivation for characteristic PKC co factors such as Ca2+/phosphatidylserine or phorbol esters strongly sug gests the involvement of this protein kinase family in regulation of N S1 replicative functions in vitro.