A novel cellular site-specific DNA-binding protein cooperates with the viral NS1 polypeptide to initiate parvovirus DNA replication

Abstract

Replication of linear single-stranded parvovirus DNA proceeds by a rolling-hairpin mechanism which generates long, palindromic, duplex concatamers. Processing to monomer length requires initiation from origins of DNA replication located at the 3 and 5 ends of each embedded monomer, reactions which can be recapitulated in vitro for minute virus of mice (MVM). To determine which cellular proteins were essential for replication from these origins, S100 extracts from 293S cells were fractionated on phosphocellulose. When recombined, these fractions were able to support replication in vitro, dependent on the viral initiator protein NS1, using plasmid forms of the 5 origin or the minimal 3 origin as templates. Fraction P-cell 1 contains two factors, replication protein A (RPA) and proliferating-cell nuclear antigen (PCNA), known to be 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 recognition 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 activated transcription factor (ATF) binding site. To identify the missing factor(s) for 3 origin replication, P-cell 1 was fractionated by further chromatography and active fractions were identified by their ability to complement RPA, PCNA, and P-cell 2 for NS1-mediated, origin-specific replication. Gel shift and UV cross-linking analysis of the replicationcompetent fractions revealed a novel 110-kDa sequence-specific DNA binding protein which recognized the consensus ATF binding site region of the origin and which we have termed parvovirus initiation factor, or PIF. Binding of PIF appears to activate the endonuclease function of NS1, allowing efficient and specific nicking of the 3 minimal origin under stringent conditions in vitro. Chemicals and reagents. Nucleotides and ribonucleotides, chelating Sepharose fast flow and Q-Sepharose were obtained from Pharmacia (Uppsala, Sweden). Poly(dI-dC), initially used as a presumptive noncompetitive inhibitor, was a double-stranded, alternating copolymer poly(dI-dC)-poly(dI-dC) and was obtained from Sigma (St. Louis, Mo.). Phosphocellulose and DE81 paper were obtained from Whatman (Springfield Mill, Maidstone, United Kingdom).Cells. Human 293S cells, obtained from Nicholas Muzyczka, University of Florida, Gainesville, Fla., were grown at 37ЊC in spinner bottles containing Joklik-modified minimal essential medium supplemented with 5% newborn calf serum. Spodoptera frugiperda (Sf9), "high five" insect cells, and recombinant baculovirus Autographa californica nuclear polyhedrosis virus were grown as previously described (9).Recombinant baculoviruses. cDNAs encoding human ATF 1, ATF 2, and ATF 3 were kindly provided by Tsonwin Hai, Ohio Biotechnology Center, Ohio State University, Columbus, Ohio, and subcloned into the baculovirus transfer vector pBlueb...

Dynamics and interactions of parvoviral NS1 protein in the nucleus

“…Proteins can be visualized as freely diffusing or interacting with nuclear chromosomes or protein compartments (Janicki and Spector, 2003;Bubulya and Spector, 2004). A number of nuclear replication proteins and APAR bodies have been shown to interact with NS1 (Christensen et al, 1997;Cziepluch et al, 2000;Bashir et al, 2001;Young et al, 2002a). However, the dynamics of this interaction has never been assessed.…”

Exploring the contribution of distal P4 promoter elements to the oncoselectivity of Minute Virus of Mice

“…The sequence ACGTCAC has been shown to bind a variety of CREB/ATF family proteins present in cell extracts (Perros et al, 1995;Christensen et al, 1997a). However, the combinations of the 20 or so members of the ATF/CREB family of bZIP transcription factors potentially able to bind the MVM CRE as homodimers or heterodimers are legion.…”

“…Recombinant human placental CREB-327 protein was expressed from a baculovirus vector (Chen et al, 1991) and purified as reported previously (Christensen et al, 1997a). Selection experiments were performed, as described by Burnett et al (2001), using oligonucleotide P1, 5′-TGACAGTTCTGACCAAGGNNNCGNNNNNNNNNNNNNCCAGGTACCTATAGTTG Gf, where underlined sequences represent binding sites for forward Primer-1, 5′-TGACAGTTCTGACCAAGG-3′, and reverse Primer-2, 5′-CCAACTATAGGTACCTGG-3′, respectively.…”

“…The CREB protein binding sequence has generally been accepted, in the literature, as the palindrome TGACGTCA (Montminy et al, 1986;Iguchi-Ariga and Schaffner, 1989), but this factor is known to be involved in the transcriptional upregulation of the retinoblastoma gene promoter, by binding to the asymmetric sequence ACGTCAC (Sakai et al, 1991;Gill et al, 1994). The MVM lefthand origin site also contains the sequence ACGTCAC and binds purified recombinant CREB and ATF1 directly and sequesters ATF/CREB family members when incubated in cell extracts (Perros et al, 1995;Christensen et al, 1997a;Burnett, 2002). This site is also known to be involved in the basal activity of the P4 promoter and to contribute to its ras transformationassociated stimulation (Perros et al, 1995).…”

Cellular Proteins Required for Adeno-Associated Virus DNA Replication in the Absence of Adenovirus Coinfection

“…Alternatively, we may have purified away a factor that is not present in fraction IIA and that stimulates AAV DNA synthesis (see below). Recently, Christensen et al (8) identified a cellular factor called parvovirus initiation factor which was present in fraction I and required for nicking of the dimer bridge and initiation of DNA synthesis from the 3Ј origin in the related parvovirus minute virus of mice. Conceivably, this or a similar factor is needed for AAV as well.…”