A NOVEL CELLULAR SITE-SPECIFIC DNA-BINDING PROTEIN COOPERATES WITH THE VIRAL NS1 POLYPEPTIDE TO INITIATE PARVOVIRUS DNA-REPLICATION

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.