Biochemical activities of minute virus of mice nonstructural protein NS1 are modulated In vitro by the phosphorylation state of the polypeptide.
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 (NS1(O)) and the native (NS1(P)) polypeptides. Biochemical analyses of replicative functions of NS1(O) 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 NS1(P). In the presence of endogenous protein kinases found in replication extracts, NS1(O) showed all functions necessary for resolution and replication of the 3' dimer bridge, indicating reactivation of NS1(O) by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1(O) was incubated with protein kinase C.
Nüesch
J P
JP
Department of Applied Tumor Virology and Institut National de la Santé et de la Recherche Médicale U375, Deutsches Krebsforschungszentrum, Heidelberg, Germany. jpf.nuesch@dkfz-heidelberg.de
Corbau
R
R
Tattersall
P
P
Rommelaere
J
J
D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008915:Minute Virus of Mice / Q000378:metabolism*; D010766:Phosphorylation; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D017931:DNA Primers; C081367:NS1 protein, minute virus of mice; D017361:Viral Nonstructural Proteins; D004265:DNA Helicases