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 (M VM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as l ate during infection. NS1 is the initiator protein for viral DNA repli cation. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; tran s 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 phosphory lation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residue s with calf intestine alkaline phosphatase, and compared the biochemic al activities of the purified un(der)phosphorylated (NS1(O)) and the n ative (NS1(P)) polypeptides. Biochemical analyses of replicative funct ions of NS1(O) revealed a severe reduction of intrinsic helicase activ ity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA](2-3) was enhanced comp ared to that of NS1P. In the presence of endogenous protein kinases fo und in replication extracts, NS1(O) showed all functions necessary for resolution and replication of the 3' dimer bridge, indicating reactiv ation of NS1(O) by rephosphorylation. Partial reactivation of the heli case activity was found as well when NS1(O) was incubated with protein kinase C.