DNA unwinding functions of minute virus of mice NS1 protein are modulated specifically by the lambda isoform of protein kinase C.
The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966-9977, 1998). The present study led to the identification of atypical PKClambda/iota as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKClambda phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKClambda phosphorylation at residue S473.
Dettwiler
S
S
Applied Tumor Virology and Institut National de la Santé et de la Recherche Médicale U375, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Rommelaere
J
J
Nüesch
J P
JP
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes; D051379:Mice; D008915:Minute Virus of Mice / Q000201:enzymology*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D000099293:Protein Kinase C-lambda
D004247:DNA; D004265:DNA Helicases; D007527:Isoenzymes; D011493:Protein Kinase C; D017361:Viral Nonstructural Proteins; D000099293:Protein Kinase C-lambda; C081367:NS1 protein, minute virus of mice