Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

Abstract

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3 was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1O was incubated with protein kinase C.

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

“…Wild-type NS1 and mutant NS1 were produced from recombinant vaccinia viruses in suspension cultures of HeLa-S3 cells and harvested at 18 h postinfection (57,60). His-tagged NS1 present in nuclear extracts was dephosphorylated, or not, with calf intestine alkaline phosphatase and purified immediately on Ni 2ϩ -NTA agarose columns (60). NS1 preparations were analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were detected by Coomassie blue staining.…”

“…NS1 was indeed found to be phosphorylated in infected cells (2,11,20,51). Moreover, recent in vitro studies have shown that HeLa cell-derived native NS1 differs from its dephosphorylated counterpart in its capacity for distinct biochemical activities involved in viral DNA replication (60).…”

“…This dependence on cofactors, together with the capacity of both fractions to phosphorylate NS1 O in vitro, strongly suggests that members of the protein kinase C (PKC) family are responsible for regulation of the NS1 replicative functions. Previous analyses of selected biochemical activities of native NS1 P versus NS1 O polypeptides have shown that the intrinsic helicase function of the viral product is strikingly dependent upon phosphorylation (60). One of the protein components necessary to rescue the replication activity of NS1 O in the kinase-free system, which was enriched in atypical PKC, was also found to reactivate the helicase function of NS1 O .…”

Vesicular Transport of Progeny Parvovirus Particles through ER and Golgi Regulates Maturation and Cytolysis

“…Metabolic labeling, purification, phosphopeptide analyses and phosphor amino acid analyses [49,68] A9 cell cultures were subjected to labeling medium for 4 h (0.1 nCi/cell of [ 32 P]. Labeled cells were harvested and the respective proteins isolated by immunoprecipitations and purified by SDS-PAGE.…”

Parvovirus interference with intracellular signalling: mechanism of PKCη activation in MVM-infected A9 fibroblasts

“…Metabolic labelling and tryptic phosphopeptide analyses were essentially performed as previously described (Nuesch et al, 1998). A9 cell cultures (10 7 cells) were infected with MVMp (30 pfu cell -1 ), kept for 24 h before being incubated for 4 h in labelling medium (complete medium lacking phosphate [Gibco/BRL] and supplemented with 0.1 nCi cell -1 of [ 32 P] orthophosphate [ICN]).…”