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 cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002-8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1(P)) supported RCR under these conditions, dephosphorylated NS1 (NS1(O)) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1(O), are enriched in protein kinase C (PKC), and, when present together, reactivate NS1(O) for replication. One of these components, containing atypical PKC, was sufficient to restore NS1(O) helicase activity. The requirement of NS1(O) reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro.
Nüesch
J P
JP
Applied Tumor Virology and Institut National de la Santé et de la Recherche Médicale U375, Deutsches Krebsforschungszentrum, Heidelberg, Germany. jpf.nuesch@dkfz-heidelberg.de
Dettwiler
S
S
Corbau
R
R
Rommelaere
J
J
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004265:DNA Helicases / Q000737:chemistry / Q000502:physiology; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008915:Minute Virus of Mice / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*
C081367:NS1 protein, minute virus of mice; D017361:Viral Nonstructural Proteins; D011493:Protein Kinase C; D004265:DNA Helicases