Concerted Mitogenic Signals Control Mammalian DNA Replication Protein Kinase C Role

ABSTRACT 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 PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ 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 PKCλ phosphorylation at residue S473.

Download Full-text

Initiation of Minute Virus of Mice DNA Replication Is Regulated at the Level of Origin Unwinding by Atypical Protein Kinase C Phosphorylation of NS1

Journal of Virology ◽

10.1128/jvi.75.13.5730-5739.2001 ◽

2001 ◽

Vol 75 (13) ◽

pp. 5730-5739 ◽

Cited By ~ 24

Author(s):

Jürg P. F. Nüesch ◽

Jesper Christensen ◽

Jean Rommelaere

Keyword(s):

Protein Kinase C ◽

Dna Replication ◽

Protein Kinase ◽

Regulatory Elements ◽

Esterification Reaction ◽

Phosphorylation Sites ◽

Minute Virus Of Mice ◽

Atypical Pkc ◽

Kinase C ◽

Minute Virus

ABSTRACT Minute virus of mice nonstructural protein NS1 is a multifunctional protein that is involved in many processes necessary for virus propagation. To perform its distinct activities in timely coordinated manner, NS1 was suggested to be regulated by posttranslational modifications, in particular phosphorylation. In fact, NS1 replicative functions are dependent on protein kinase C (PKC) phosphorylation, most likely due to alteration of the biochemical profile of the viral product as determined by comparing native NS1 with its dephosphorylated counterpart. Through the characterization of NS1 mutants at individual PKC consensus phosphorylation sites for their biochemical activities and nickase function, we were able to identify two target atypical PKC phosphorylation sites, T435 and S473, serving as regulatory elements for the initiation of viral DNA replication. Furthermore, by dissociating the energy-dependent helicase activity from the ATPase-independent trans esterification reaction using partially single-stranded substrates, we could demonstrate that atypical PKC regulation of NS1 nickase activity occurs at the level of origin unwinding prior to trans esterification.

Download Full-text

Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

Journal of Virology ◽

10.1128/jvi.72.12.9966-9977.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 9966-9977 ◽

Cited By ~ 23

Author(s):

Jürg P. F. Nüesch ◽

Sabine Dettwiler ◽

Romuald Corbau ◽

Jean Rommelaere

Keyword(s):

Protein Kinase C ◽

Dna Replication ◽

Protein Kinase ◽

Protein Kinases ◽

Viral Dna ◽

Minute Virus Of Mice ◽

Viral Dna Replication ◽

Kinase C ◽

Minute Virus

ABSTRACT 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 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O 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.

Download Full-text