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 protei n involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the h ost cell. Since NS1 becomes phosphorylated during infection, it was propose d that the different tasks of this protein might be regulated in a coordina ted manner by phosphorylation. Indeed, comparing biochemical functions of n ative NS1 with its dephosphorylated counterpart showed that site-specific n icking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the prot ein to the origin became enhanced. As a consequence, the dephosphorylated p olypeptide is deficient for initiation of DNA replication. By adding fracti onated cell extracts to a kinase-free in vitro replication system, the comb ination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosph orylated NS1 protein upon addition of PKC cofactors. One of these component s, 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. Nuesch, S. Dettwi ler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966-9977, 1998). The prese nt study led to the identification of atypical PKC lambda/iota as the activ e component of HA-I responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKC lambda 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 regula tion, suggesting a possible modulation of NS1 functions by PKC lambda phosp horylation at residue S473.