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Parvoviral rolling hairpin replication generates palindromic genomic concatemers whose junctions are resolved to give unit-length genomes by a process involving DNA replication initiated at origins derived from each viral telomere. The left-end origin of minute virus of mice (MVM), oriL, contains binding sites for the viral initiator nickase, NS1, and parvovirus initiation factor (PIF), a member of the emerging KDWK family of transcription factors. oriL is generated as an active form, oriL(TC), and as an inactive form, oriL(GAA), which contains a single additional nucleotide inserted between the NS1 and PIF sites. Here we examined the interactions on oriL(TC) which lead to activation of NS1 by PIF. The two subunits of PIF, p79 and p96, cooperatively bind two ACGT half-sites, which can be flexibly spaced. When coexpressed from recombinant baculoviruses, the PIF subunits preferentially form heterodimers which, in the presence of ATP, show cooperative binding with NS1 on oriL, but this interaction is preferentially enhanced on oriL(TC) compared to oriL(GAA). Without ATP, NS1 is unable to bind stably to its cognate site, but PIF facilitates this interaction, rendering the NS1 binding site, but not the nick site, resistant to DNase I. Varying the spacing of the PIF half-sites shows that the distance between the NS1 binding site and the NS1-proximal half-site is critical for nickase activation, whereas the position of the distal half-site is unimportant. When expressed separately, both PIF subunits form homodimers that bind site specifically to oriL, but only complexes containing p79 activate the NS1 nickase function.

Mutants of minute virus of mice (MVM) which express truncated forms of the NS2 polypeptide are known to exhibit a host range defect, replicating productively in transformed human cells but not in cells from their normal murine host. To explore this deficiency we generated viruses with translation termination codons at various positions in the second exon of NS2. In human cells these mutants were viable, but showed a late defect in progeny virion release which put them at a selective disadvantage compared to the wildtype. In murine cells, however, duplex viral DNA amplification was reduced to 5% of wildtype levels and single-strand DNA synthesis was undetectable. These deficiencies could not be attributed to a failure to initiate infection or to a generalized defect in viral gene expression, since the viral replicator protein NS1 was expressed to normal or elevated levels early in infection. In contrast, truncated NS2 gene products failed to accumulate, so that each mutant exhibited a similar NS2-null phenotype. Expression of the capsid polypeptides VP1 and VP2 and their subsequent assembly into intact particles were examined in detail. Synchronized infected cell populations labeled under pulse-chase conditions were analyzed by differential immunoprecipitation of native or denatured extracts using antibodies which discriminated between intact particles and isolated polypeptide chains. These analyses showed that at early times in infection, capsid protein synthesis and stability were normal, but particle assembly was impaired. Unassembled VP proteins were retained in the cell for several hours, but as the unprocessed material accumulated, capsid protein synthesis progressively diminished, so that at later times relatively few VP molecules were synthesized. Thus in NS2-null infections of mouse cells there is a major primary defect in the folding or assembly processes required for effective capsid production.

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 replication-competent 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.

The NS1 polypeptide of minute virus of mice (MVM) is a potent transcriptional activator of the MVM P38 promoter. The minimum region of this promoter required for transactivation has been identified and termed the transactivation region (tar). However, the function of tar and the biochemical steps involved in NS1-mediated transactivation are not well understood. Here we provide evidence that NS1 binds directly and specifically to tar in a strictly ATP-dependent manner. A DNA fragment containing tar was specifically coimmunoprecipitated with purified baculovirus-expressed MVM NS1, using antibodies directed against NS1 amino- or carboxy-terminal peptides. Using this immunoprecipitation assay, we found that the NS1-tar interaction was enhanced approximately 10-fold by ATP, but subsequent incubation at elevated temperatures in the presence, but not the absence, of MgCl2 caused rapid loss of tar binding. This finding suggests that the tar-NS1 complex has a short half-life under assay conditions which favor ATP hydrolysis. Specific binding was efficiently inhibited by self-ligated oligonucleotides containing the core DNA sequence (ACCA)3, but the same nonligated 20- and 21-mer oligonucleotides were unable to compete effectively, indicating that NS1 only binds to its cognate site when this site is presented on DNA fragments of sufficient size. DNase I footprinting experiments performed in the presence of gamma S-ATP revealed that NS1 protects a 43-bp sequence extending asymmetrically from the (ACCA)2 sequence toward the TATA box of the promoter. NS1 footprints obtained at other sites in the MVM genome were similarly large and asymmetric, all extending approximately 31 bp 5' from the core (ACCA)2-3 sequence. Surprisingly, no footprints were obtained in the absence of gamma S-ATP even under low-stringency binding conditions. However, ATP could be omitted from the reactions if NS1 was first incubated with antibodies directed against its 16-amino-acid carboxy-terminal peptide. Since these antibodies probably create intermolecular cross-links, this finding suggests that NS1 may only bind its cognate site efficiently, or perhaps at all, if the transactivator is first induced to form oligomers. From these data, we hypothesize that ATP binding may also induce NS1 to oligomerize and that such assembly is required before the protein can bind effectively to the tar sequence. The functional implications of the NS1-tar interaction will be discussed.

Parvoviral DNA replication has many features in common with prokaryotic rolling circle replication (RCR), including the pivotal role of an initiator protein which introduces a site-specific, single strand nick into a duplex origin sequence. In this process, the protein becomes covalently attached to the new 5' end of the DNA, while making available a 3' hydroxyl to prime de novo synthesis. Sequence comparisons of prokaryotic RCR initiators has revealed a set of three common motifs, two of which, a putative metal coordination site and a downstream active-site tyrosine motif, could be tentatively identified in parvoviral replicator proteins. We have introduced mutations into the NS1 gene of the murine parvovirus minute virus of mice (MVM), in the putative metal coordination site at H129, and into the three candidate tyrosine motifs at Y188, Y197, and Y210. Histidine-tagged mutant proteins were expressed in HeLa cells from recombinant vaccinia virus vectors and partially purified. None of the mutant proteins were able to initiate replication of origin-containing plasmids in vitro, and each showed impaired site-specific binding to the viral origin, with Y188 and Y197 being most severely defective. If this deficiency was minimized using low salt conditions, however, Y188 and Y197 mutant proteins were able to nick and become covalently attached to origin DNA, whereas Y210 and H129 mutant proteins were not, suggesting that the latter residues are part of the catalytic site of the NS1 nickase. Transfer of [32P]phosphate from substrate DNA to NS1, followed by cyanogen bromide cleavage of the complex, gave the single, labeled peptide consistent with Y210 being the linking tyrosine.

A DNA fragment containing the minute virus of mice 3' replication origin was specifically coprecipitated in immune complexes containing the virally coded NS1, but not the NS2, polypeptide. Antibodies directed against the amino- or carboxy-terminal regions of NS1 precipitated the NS1-origin complexes, but antibodies directed against NS1 amino acids 284 to 459 blocked complex formation. Using affinity-purified histidine-tagged NS1 preparations, we have shown that the specific protein-DNA interaction is of moderate affinity, being stable in 0.1 M salt but rapidly lost at higher salt concentrations. In contrast, generalized (or nonspecific) DNA binding by NS1 could be demonstrated only in low salt. Addition of ATP or gamma S-ATP enhanced specific DNA binding by wild-type NS1 severalfold, but binding was lost under conditions which favored ATP hydrolysis. NS1 molecules with mutations in a critical lysine residue (amino acid 405) in the consensus ATP-binding site bound to the origin, but this binding could not be enhanced by ATP addition. DNase I protection assays carried out with wild-type NS1 in the presence of gamma S-ATP gave footprints which extended over 43 nucleotides on both DNA strands, from the middle of the origin bubble sequence to a position some 14 bp beyond the nick site. The DNA-binding site for NS1 was mapped to a 22-bp fragment from the middle of the 3' replication origin which contains the sequence ACCAACCA. This conforms to a reiterated motif (ACCA)2-3, which occurs, in more or less degenerate form, at many sites throughout the minute virus of mice genome (J. W. Bodner, Virus Genes 2:167-182, 1989). Insertion of a single copy of the sequence (ACCA)3 was shown to be sufficient to confer NS1 binding on an otherwise unrecognized plasmid fragment. The functions of NS1 in the viral life cycle are reevaluated in the light of this result.

The gene encoding the major replicative protein, NS1, of minute virus of mice (MVM) was transferred into a recombinant vaccinia virus vector in place of the vaccinia thymidine kinase gene. The NS1 gene was placed under control of a bacteriophage T7 promoter and expressed in cells coinfected with another recombinant vaccinia virus, vTF7-3, which encodes the T7 RNA polymerase. Expression of NS1 was further enhanced by the presence of a 5' untranslated region, derived from encephalomyocarditis virus, which allows efficient cap-independent translation. This system was used to produce and analyze wild-type NS1 and two mutant forms of the protein, NS1K405R and NS1K405M, in which the highly conserved lysine codon located in the putative purine triphosphate binding site of NS1 was changed to arginine and methionine, respectively. Full-length NS1 was expressed efficiently in both human and mouse cells infected with each of the three recombinant viruses, and in each case the NS1 was rapidly and efficiently translocated into the nucleus. Wild-type NS1 expressed in this way was biologically active. It was able to trans-activate an MVM P38 promoter located in a host chromosomal site, whereas the two mutant forms of NS1 showed no significant activity in this assay, and it was capable of resolving palindromic junction fragments cloned from multimeric MVM replicative form DNA molecules. These substrates, representing MVM genomic left-end:left-end and right-end:right-end fusions, were resolved in a DNA synthesis-dependent in vitro reaction supplemented with nuclear extracts containing recombinant wild-type NS1. Neither of the two mutant forms of the polypeptide had any detectable activity in this assay.