J. Christensen et al., A NOVEL CELLULAR SITE-SPECIFIC DNA-BINDING PROTEIN COOPERATES WITH THE VIRAL NS1 POLYPEPTIDE TO INITIATE PARVOVIRUS DNA-REPLICATION, Journal of virology, 71(2), 1997, pp. 1405-1416
Replication of linear single-stranded parvovirus DNA proceeds by a rol
ling-hairpin mechanism which generates long, palindromic, duplex conca
tamers. Processing to monomer length requires initiation from origins
of DNA replication located at the 3' and 5' ends of each embedded mono
mer, reactions which can be recapitulated in vitro for minute virus of
mice (MVM). To determine which cellular proteins were essential for r
eplication from these origins, S100 extracts from 293S cells were frac
tionated on phosphocellulose. When recombined, these fractions were ab
le to support replication in vitro, dependent on the viral initiator p
rotein NS1, using plasmid forms of the 5' origin or the minimal 3' ori
gin as templates. Fraction P-cell 1 contains two factors, replication
protein A (RPA) and proliferating-cell nuclear antigen (PCNA), known t
o he essential for simian virus 40 replication in vitro. When P-cell 1
was replaced with purified recombinant RPA and PCNA, NS1-mediated MVM
replication initiated from the 5' origin but not from the 3' origin.
The 3' origin is a 50-bp sequence containing three distinct recognitio
n elements, an NS1 binding site, a site at which NS1 nicks the DNA to
generate the priming 3' OH, and a region containing a consensus activa
ted transcription factor (ATF) binding site. To identify the missing f
actor(s) for 3' origin replication, P-cell 1 was fractionated by furth
er chromatography and active fractions were identified by their abilit
y to complement RPA, PCNA, and P-cell 2 for NS1-mediated, origin-speci
fic replication. Gel shift and UV cross-linking analysis of the replic
ation-competent fractions revealed a novel 110-kDa sequence-specific D
NA binding protein which recognized the consensus ATF binding site reg
ion of the origin and which we have termed parvovirus initiation facto
r, or PIF. Binding of PIF appears to activate the endonuclease functio
n of NS1, allowing efficient and specific nicking of the 3' minimal or
igin under stringent conditions in vitro.