Jpf. Nuesch et al., BIOCHEMICAL ACTIVITIES OF MINUTE VIRUS OF MICE NONSTRUCTURAL PROTEIN NS1 ARE MODULATED IN-VITRO BY THE PHOSPHORYLATION STATE OF THE POLYPEPTIDE, Journal of virology, 72(10), 1998, pp. 8002-8012
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.