The left-end and right-end origins of minute virus of mice DNA differ in their capacity to direct episomal amplification and integration in vivo.

Previously it was shown that a 53-nucleotide viral replication origin, derived from the left-end (3') telomere of minute virus of mice (MVM) DNA, directed integration of infecting MVM genomes into an Epstein-Barr virus (EBV)-based episome in cell culture. Integration depended upon the presence, in the episome, of a functional origin sequence which could be nicked by NS1, the viral initiator protein. Here we extend our studies to the genomic right-end (5') origin and report that three 131- to 135-nucleotide right-end origin sequences failed to target MVM episomal integration even though the same sequences were functional in NS1-driven DNA replication assays in vitro. Additionally, we observed amplification of episomal DNA in response to MVM infection in cell lines harboring episomes which directed integration, but not in cell lines containing episomes which did not direct integration, including those with inserts of the MVM right-end origin.

Minute virus of mice initiator protein NS1 and a host KDWK family transcription factor must form a precise ternary complex with origin DNA for nicking to occur.

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.

Two widely spaced initiator binding sites create an HMG1-dependent parvovirus rolling-hairpin replication origin.

Minute virus of mice (MVM) replicates via a linearized form of rolling-circle replication in which the viral nickase, NS1, initiates DNA synthesis by introducing a site-specific nick into either of two distinct origin sequences. In vitro nicking and replication assays with substrates that had deletions or mutations were used to explore the sequences and structural elements essential for activity of one of these origins, located in the right-end (5') viral telomere. This structure contains 248 nucleotides, most-favorably arranged as a simple hairpin with six unpaired bases. However, a pair of opposing NS1 binding sites, located near its outboard end, create a 33-bp palindrome that could potentially assume an alternate cruciform configuration and hence directly bind HMG1, the essential cofactor for this origin. The palindromic nature of this sequence, and thus its ability to fold into a cruciform, was dispensable for origin function, as was the NS1 binding site occupying the inboard arm of the palindrome. In contrast, the NS1 site in the outboard arm was essential for initiation, even though positioned 120 bp from the nick site. The specific sequence of the nick site and an additional NS1 binding site which directly orients NS1 over the initiation site were also essential and delimited the inboard border of the minimal right-end origin. DNase I and hydroxyl radical footprints defined sequences protected by NS1 and suggest that HMG1 allows the NS1 molecules positioned at each end of the origin to interact, creating a distortion characteristic of a double helical loop.

Two new members of the emerging KDWK family of combinatorial transcription modulators bind as a heterodimer to flexibly spaced PuCGPy half-sites.

Initially recognized as a HeLa factor essential for parvovirus DNA replication, parvovirus initiation factor (PIF) is a site-specific DNA-binding complex consisting of p96 and p79 subunits. We have cloned and sequenced the human cDNAs encoding each subunit and characterized their products expressed from recombinant baculoviruses. The p96 and p79 polypeptides have 40% amino acid identity, focused particularly within a 94-residue region containing the sequence KDWK. This motif, first described for the Drosophila homeobox activator DEAF-1, identifies an emerging group of metazoan transcriptional modulators. During viral replication, PIF critically regulates the viral nickase, but in the host cell it probably modulates transcription, since each subunit is active in promoter activation assays and the complex binds to previously described regulatory elements in the tyrosine aminotransferase and transferrin receptor promoters. Within its recognition site, PIF binds coordinately to two copies of the tetranucleotide PuCGPy, which, remarkably, can be spaced from 1 to 15 nucleotides apart, a novel flexibility that we suggest may be characteristic of the KDWK family. Such tetranucleotides are common in promoter regions, particularly in activating transcription factor/cyclic AMP response element-binding protein (ATF/CREB) and E-box motifs, suggesting that PIF may modulate the transcription of many genes.

cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses.

The replication of viral genomes and the production of recombinant viral vectors from infectious molecular clones of parvoviruses MVMp and H1 were greatly improved by the introduction of a consensus NS-1 nick site at the junction between the left-hand viral terminus and the plasmid DNA. Progressive deletions of up to 1600 bp in the region encoding the structural genes as well as insertions of foreign DNA in replacement of those sequences did not appreciably affect the replication ability of the recombinant H1 virus genomes. In contrast, the incorporation of these genomes into recombinant particles appeared to depend on in cis-provided structural gene sequences. Indeed, the production of H1 viral vectors by cotransfection of recombinant clones and helper plasmids providing the structural proteins (VPs) in trans, drastically decreased when more than 800 bp was removed from the VP transcription unit. Furthermore, titers of viral vectors, in which most of the VP-coding region was replaced by an equivalent-length sequence consisting of reporter cDNA and stuffer DNA, were reduced more than 50 times in comparison with recombinant vectors in which stuffer DNA was not substituted for the residual VP sequence. In addition, viral vector production was restricted by the overall size of the genome, with a mere 6% increase in DNA length leading to an approximately 10 times lower encapsidation yield. Under conditions fulfilling the above-mentioned requirements for efficient packaging, titers of virus vectors from improved recombinant molecular DNA clones amounted to 5 x 10(7) infectious units per milliliter of crude extract. These titers should allow the assessment of the therapeutic effect of recombinant parvoviruses expressing small transgenes in laboratory animals.

Controlled conformational transitions in the MVM virion expose the VP1 N-terminus and viral genome without particle disassembly.

Antisera were raised against peptides corresponding to the N-termini of capsid proteins VP1 and VP2 from the parvovirus minute virus of mice. Epitopes in the 142-amino-acid VP1-specific region were not accessible in the great majority of newly released viral particles, and sera directed against them failed to neutralize virus directly or deplete stocks of infectious virions. However, brief exposure to temperatures of 45 degreesC or more induced a conformational transition in a population of full virions, but not in empty viral particles, in which VP1-specific sequences became externally accessible. In contrast, the VP2 N-terminus was antibody-accessible in all full, but not empty, particles without prior treatment. An electrophoretic mobility shift assay, in which particles were heat-treated and/or preincubated with antibodies prior to electrophoresis, confirmed this pattern of epitope accessibility, showing that the heat-induced conformational transition produces a retarded form of virion that can be supershifted by incubation with VP1-specific sera. The proportion of virions undergoing transition increased with temperature, but at all temperatures up to 70 degreesC viral particles retained structure-specific antigenic determinants and remained essentially intact, without shedding individual polypeptide species or subunits. However, despite the apparent integrity of its protective coat, the genome became accessible to externally applied enzymes in an increasing proportion of virions through this temperature range, suggesting that the conformational transitions that expose VP1 likely also allow access to the genome. Heating particles to 80 degreesC or above finally induced disassembly to polypeptide monomers.

High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin.

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.

Parvovirus initiation factor PIF: a novel human DNA-binding factor which coordinately recognizes two ACGT motifs.

A novel human site-specific DNA-binding factor has been partially purified from extracts of HeLa S3 cells. This factor, designated PIF, for parvovirus initiation factor, binds to the minimal origin of DNA replication at the 3' end of the minute virus of mice (MVM) genome and functions as an essential cofactor in the replication initiation process. Here we show that PIF is required for the viral replicator protein NS1 to nick and become covalently attached to a specific site in the origin sequence in a reaction which requires ATP hydrolysis. DNase I and copper ortho-phenanthroline degradation of the PIF-DNA complexes showed that PIF protects a stretch of some 20 nucleotides, covering the entire region in the minimal left-end origin not already known to be occupied by NS1. Methylation and carboxy-ethylation interference analysis identified two ACGT motifs, spaced by five nucleotides, as the sequences responsible for this binding. A series of mutant oligonucleotides was then used as competitive inhibitors in gel mobility shift assays to confirm that PIF recognizes both of these ACGT sequences and to demonstrate that the two motifs comprise a single binding site rather than two separate sites. Competitive inhibition of the origin nicking assay, using the same group of oligonucleotides, confirmed that the same cellular factor is responsible for both mobility shift and nicking activities. UV cross-linking and relative mobility assays suggest that PIF binds DNA as a heterodimer or higher-order multimer with subunits in the 80- to 100-kDa range.

The NS2 polypeptide of parvovirus MVM is required for capsid assembly in murine cells.

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.

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 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.

Minute virus of mice transcriptional activator protein NS1 binds directly to the transactivation region of the viral P38 promoter in a strictly ATP-dependent manner.

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.

Sequence motifs in the replicator protein of parvovirus MVM essential for nicking and covalent attachment to the viral origin: identification of the linking tyrosine.

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.

The NS1 polypeptide of the murine parvovirus minute virus of mice binds to DNA sequences containing the motif [ACCA]2-3.

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.

An asymmetric nucleotide in the parvoviral 3' hairpin directs segregation of a single active origin of DNA replication.

The 3' telomere of the linear single-stranded DNA genome of minute virus of mice (MVM), a murine parvovirus, can assume a complex hairpin structure. This contains a stem in which there is a mismatched 'bubble' sequence where a GA doublet opposes a GAA triplet. During replication, this hairpin is copied to form an imperfect palindrome which bridges adjacent genomes in a dimer duplex intermediate, leaving the two 'bubble' sequences embedded in potential replication origins on either side of the axis of symmetry. Such junctions are resolved asymmetrically in vitro in a DNA synthetic reaction which requires the viral initiator protein NS1. We show that the sequence surrounding the doublet is a potent origin, but the analogous region containing the triplet is completely inactive. The active origin is approximately 50 bp long, extending from an Activated Transcription Factor binding site at one end to a position some 7 bp beyond the major initiation site, to which NS1 ultimately becomes covalently attached. The actual sequence of the GA doublet is unimportant, but insertion of any third nucleotide here inactivates the origin, indicating that it represents a critical spacer element. Segregation of this asymmetry, therefore, allows the virus to confine replication initiation to one particular telomeric configuration.

Asymmetric resolution of a parvovirus palindrome in vitro.

Cell extracts from murine A9 or human HeLa cells containing wild-type copies the NS1 polypeptide of minute virus of mice (MVM), produced from a recombinant vaccinia virus, can support the resolution of viral 3' termini from palindromic junction fragments of dimeric, replicative-form MVM DNA. Resolution resulted in the generation of two new viral termini, one associated with each arm of the junction palindrome. Telomeres were created in two configurations, "extended" forms, which were covalently associated with NS1 molecules, and smaller "turn-around" forms in which a single arm of the palindrome terminated at the axis of dyad symmetry in a covalent bond which cross-linked the two strands. The in vitro resolution reaction was asymmetric, generating predominantly extended-form termini from one arm of the palindrome but predominantly turn-around forms from the other. This asymmetry was independent of the type of cell used to prepare the in vitro extract or the orientation of the palindrome in the plasmid and was obtained for all cloned junction sequences of 156 bp or more. Two modified forms of the duplex junction fragment, which appeared to be intermediates in the resolution process since they were nicked, covalently linked to NS1, and associated with newly synthesized DNA, were identified. The structures of these intermediates suggest that resolution is initiated by preferential nicking at one of the two candidate resolution sites. The asymmetric nature of this resolution reaction is discussed in terms of current models of MVM DNA replication.

Expression of functional parvoviral NS1 from recombinant vaccinia virus: effects of mutations in the nucleotide-binding motif.

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.

In vitro excision and replication of 5' telomeres of minute virus of mice DNA from cloned palindromic concatemer junctions.

HeLa cell extracts containing wild-type copies of the minute virus of mice NS-1 polypeptide produced from a recombinant vaccinia virus vector could support the excision and replication of viral 5' telomeres from cloned concatemer junction fragments. Resolution did not occur if wild-type NS-1 was omitted from the extract or if the substrate DNA contained palindromic sequences without specific viral resolution sites. In the presence of NS-1, [32P]dGTP incorporation into all templates was slightly increased, but if the template contained specific viral resolution sites DNA synthesis was greatly enhanced, and took two distinct forms: (i) generation of a limited number of high-molecular-weight molecules, probably due to a form of rolling-circle replication, and (ii) synthesis of new DNA at the viral telomeres. Resolution of the junction fragment generated two newly synthesized viral telomeres, each of which was covalently associated with NS-1 and contained a duplex copy of the complex palindrome located around the axis of symmetry of the concatemer junction. Cloned junction fragments of 296 bp or more could be resolved efficiently in vitro, and since NS-1 molecules were left covalently attached to the newly resolved termini, the latter could be partially purified by immuno-precipitation with anti-NS-1 serum. Restriction analysis and further fractionation of the precipitated DNA showed that the in vitro resolution sites were in the predicted positions on either side of the axis of symmetry, and that de novo DNA synthesis was associated predominantly with one of the two daughter strands. Telomeres were generated from both arms of the substrate with equal efficiency, and contained the characteristic "flip" and "flop" sequence inversions observed in vivo. Since a high proportion of termini were associated with adjacent viral sequences that retained the bacterial methylation pattern, in vitro resolution was not dependent upon prior DNA replication proceeding through the entire palindromic insert.

In vivo resolution of circular plasmids containing concatemer junction fragments from minute virus of mice DNA and their subsequent replication as linear molecules.

During replication of their linear, single-stranded DNA genomes, parvoviruses generate a series of concatemeric duplex intermediates. We have cloned, into Escherichia coli plasmids, junction fragments from these palindromic concatemers of minute virus of mice DNA spanning both the right end-to-right end (viral 5' to 5') and left end-to-left end (viral 3' to 3') fusions. When mouse cells were transfected with these circular plasmids and superinfected with minute virus of mice, the viral junctions were resolved and the plasmids replicated as linear chromosomes with vector DNA in their centers and viral DNA at their termini. Resolution did not occur when the concatemer joint was replaced by a different palindromic sequence or when the transfected cells were not superinfected, indicating the presence of latent origins of replication which could only be activated by a viral trans-acting factor(s). Moreover, the products of resolution and replication from the two termini were characteristically different. Analysis of individual terminal fragments showed that viral 5' (right-end) sequences were resolved predominantly into "extended" structures with covalently associated copies of the virally encoded NS-1 polypeptide, while bridges derived from the 3' (left) end resolved into both NS-1-associated extended termini and lower-molecular-weight "turn-around" forms in which the two DNA strands were covalently continuous. This pattern of resolution exactly coincides with that seen at the two termini of replicative-form intermediates in normal virus infections. These results demonstrate that the bridge structures are authentic substrates for resolution and indicate that the frequency with which extended versus turn-around forms of each terminus are generated is an intrinsic property of the telomere.

Alternate splicing in a parvoviral nonstructural gene links a common amino-terminal sequence to downstream domains which confer radically different localization and turnover characteristics.

Minute virus of mice (MVM) encodes two groups of nonstructural proteins, the 83-kDa NS-1 polypeptides encoded from a contiguous sequence in the left half of the genome and the 25-kDa NS-2 polypeptides, which share a common amino-terminal domain with NS-1 but are multiply spliced. Peptide-specific antibodies were used to demonstrate that three alternatively spliced forms of NS-2 are synthesized when synchronized A9 cells are infected with the prototype strains of MVM, MVM(p), and that each of these species migrates as two bands on sodium dodecyl sulfate-gel electrophoresis, due to the presence of both phosphorylated and unphosphorylated forms. While most NS-1 molecules are located in the nucleus, all three species of NS-2 are predominantly cytoplasmic, and their phosphorylated forms are exclusively cytoplasmic. Although both NS-1 and NS-2 molecules are synthesized early in infection, all forms of NS-2 are synthesized and accumulate three to four times as NS-1 molecules, making them the predominant virally coded proteins in the cell at this time. Despite their common amino-terminal domain, NS-2 molecules turn over rapidly while NS-1 polypeptides persist for many hours. Apart from the fact that the three NS-2 gene products are synthesized in different molar amounts, we were unable to detect any differences in the expression, stability, distribution, or phosphorylation of the various molecular forms, suggesting that these latter characteristics are mediated by their common internal exon.

Susceptibility of human cells to killing by the parvoviruses H-1 and minute virus of mice correlates with viral transcription.

Human fibroblasts and epithelial cells differing in their susceptibility to killing by the autonomous parvoviruses H-1 and minute virus of mice were compared for their capacity to express viral mRNAs and proteins. The transition from a parvovirus-resistant to a parvovirus-sensitive phenotype correlated with a proportional increase in the production of the three major viral transcripts and of structural and nonstructural proteins. In contrast, cell sensitization to parvovirus could not be correlated with detectable changes in virus uptake, intracellular localization of gene products, stability of viral mRNAs, or phosphorylation of viral nonstructural polypeptides. Moreover, the H-1 virus-sensitive keratinocyte line studied did not sustain a greater level of viral DNA amplification than its resistant derivative. Therefore, the differential susceptibility of the human cells tested to parvovirus infection appears to be mainly controlled at the level of transcription of the viral genome. Parvoviral gene expression could not be elevated by increasing the input multiplicity of infection in either of the cell systems analyzed. Together, these data suggest that a cellular factor(s) regulating parvoviral transcription may be modulated by oncogenic transformation or by differentiation, as both features have been shown to affect cell susceptibility to parvoviruses.