The major nonstructural protein of parvovirus MVMp, NS1, is an 83-kDa nucle
ar phosphoprotein which exerts a variety of functions during a viral infect
ion. These multiple tasks range from its major involvement in viral DNA amp
lification and promoter regulation to the cytotoxic action on the host cell
. Since these most divergent functions are exerted in an orderly fashion, i
t has been proposed that NS1 is regulated by posttranslational modification
s, in particular phosphorylation. So far it has been shown that the capacit
y of NS1 for initiation of replication is regulated in vitro by phosphoryla
tion through members of the protein kinase C family, most likely as a resul
t of control of the DNA unwinding activity (J. P. F. Nuesch et al., 1998, J
. Virol. 72, 9966-9977). To substantiate these in vitro findings in vivo, w
e investigated NS1 phosphorylation during an MVMp infection in a natural ho
st cell, A9 fibroblasts, with reference to characteristic features of the v
irus cycle. The NS1 phosphorylation pattern was found to change throughout
the infection, raising the possibility that distinct tasks of NS1 might be
achieved through differential phosphorylation of the polypeptide. In additi
on, we present in vivo evidence that a phosphorylated form of NS1 is able t
o initiate viral DNA replication and becomes covalently attached to replica
ted DNA. Moreover, NS1 was found to be phosphorylated in vivo within the he
licase domain. showing alignment with at least one phosphopeptide generated
by an "activating" kinase in vitro. These data suggest that phosphorylatio
n-mediated regulation of NS1 for replicative functions as observed in vitro
may also take place during a natural virus infection. (C) 1999 Academic Pr
ess.