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Parvoviridae, a diverse family of small single-stranded DNA viruses was established in 1975. It was divided into two subfamilies, Parvovirinae and Densovirinae, in 1993 to accommodate parvoviruses that infect vertebrate and invertebrate animals, respectively. This relatively straightforward segregation, using host association as the prime criterion for subfamily-level classification, has recently been challenged by the discovery of divergent, vertebrate-infecting parvoviruses, dubbed "chapparvoviruses", which have proven to be more closely related to viruses in certain Densovirinae genera than to members of the Parvovirinae. Viruses belonging to these genera, namely Brevi-, Hepan- and Penstyldensovirus, are responsible for the unmatched heterogeneity of the subfamily Densovirinae when compared to the Parvovirinae in matters of genome organization, protein sequence homology, and phylogeny. Another genus of Densovirinae, Ambidensovirus, has challenged traditional parvovirus classification, as it includes all newly discovered densoviruses with an ambisense genome organization, which introduces genus-level paraphyly. Lastly, current taxon definition and virus inclusion criteria have significantly limited the classification of certain long-discovered parvoviruses and impedes the classification of some potential family members discovered using high-throughput sequencing methods. Here, we present a new and updated system for parvovirus classification, which includes the introduction of a third subfamily, Hamaparvovirinae, resolves the paraphyly within genus Ambidensovirus, and introduces new genera and species into the subfamily Parvovirinae. These proposals were accepted by the ICTV in 2020 March.

Members of the family Parvoviridae are small, resilient, non-enveloped viruses with linear, single-stranded DNA genomes of 4-6 kb. Viruses in two subfamilies, the Parvovirinae and Densovirinae, are distinguished primarily by their respective ability to infect vertebrates (including humans) versus invertebrates. Being genetically limited, most parvoviruses require actively dividing host cells and are host and/or tissue specific. Some cause diseases, which range from subclinical to lethal. A few require co-infection with helper viruses from other families. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Parvoviridae, which is available at www.ictv.global/report/parvoviridae.

Motivation: The increasing rate of submission of genetic sequences into public databases is providing a growing resource for classifying the organisms that these sequences represent. To aid viral classification, we have developed ViCTree, which automatically integrates the relevant sets of sequences in NCBI GenBank and transforms them into an interactive maximum likelihood phylogenetic tree that can be updated automatically. ViCTree incorporates ViCTreeView, which is a JavaScript-based visualization tool that enables the tree to be explored interactively in the context of pairwise distance data., Results: To demonstrate utility, ViCTree was applied to subfamily Densovirinae of family Parvoviridae. This led to the identification of six new species of insect virus., Availability and Implementation: ViCTree is open-source and can be run on any Linux- or Unix-based computer or cluster. A tutorial, the documentation and the source code are available under a GPL3 license, and can be accessed at http://bioinformatics.cvr.ac.uk/victree_web/., Supplementary Information: Supplementary data are available at Bioinformatics online.

Combining virus-enhanced immunogenicity with direct delivery of immunomodulatory molecules would represent a novel treatment modality for melanoma, and would require development of new viral vectors capable of targeting melanoma cells preferentially. Here we explore the use of rodent protoparvoviruses targeting cells of the murine melanoma model B16F10. An uncloned stock of mouse parvovirus 1 (MPV1) showed some efficacy, which was substantially enhanced following serial passage in the target cell. Molecular cloning of the genes of both starter and selected virus pools revealed considerable sequence diversity. Chimera analysis mapped the majority of the improved infectivity to the product of the major coat protein gene, VP2 , in which linked blocks of amino acid changes and one or other of two apparently spontaneous mutations were selected. Intragenic chimeras showed that these represented separable components, both contributing to enhanced infection. Comparison of biochemical parameters of infection by clonal viruses indicated that the enhancement due to changes in VP2 operates after the virus has bound to the cell surface and penetrated into the cell. Construction of an in silico homology model for MPV1 allowed placement of these changes within the capsid shell, and revealed aspects of the capsid involved in infection initiation that had not been previously recognized., Competing Interests: The authors declare no conflict of interest.

In minute virus of mice (MVM) capsids, icosahedral five-fold channels serve as portals mediating genome packaging, genome release, and the phased extrusion of viral peptides. Previous studies suggest that residues L172 and V40 are essential for channel function. The structures of MVMi wildtype, and mutant L172T and V40A virus-like particles (VLPs) were solved from cryo-EM data. Two constriction points, termed the mid-gate and inner-gate, were observed in the channels of wildtype particles, involving residues L172 and V40 respectively. While the mid-gate of V40A VLPs appeared normal, in L172T adjacent channel walls were altered, and in both mutants there was major disruption of the inner-gate, demonstrating that direct L172:V40 bonding is essential for its structural integrity. In wildtype particles, residues from the N-termini of VP2 map into claw-like densities positioned below the channel opening, which become disordered in the mutants, implicating both L172 and V40 in the organization of VP2 N-termini., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Parvoviruses are small, rugged, nonenveloped protein particles containing a linear, nonpermuted, single-stranded DNA genome of ∼5 kb. Their limited coding potential requires optimal adaptation to the environment of particular host cells, where entry is mediated by a variable program of capsid dynamics, ultimately leading to genome ejection from intact particles within the host nucleus. Genomes are amplified by a continuous unidirectional strand-displacement mechanism, a linear adaptation of rolling circle replication that relies on the repeated folding and unfolding of small hairpin telomeres to reorient the advancing fork. Progeny genomes are propelled by the viral helicase into the preformed capsid via a pore at one of its icosahedral fivefold axes. Here we explore how the fine-tuning of this unique replication system and the mechanics that regulate opening and closing of the capsid fivefold portals have evolved in different viral lineages to create a remarkably complex spectrum of phenotypes.

The orphan parvovirus tumor virus X (TVX) has potent oncolytic activity. Compared to other viruses from the species Rodent protoparvovirus 1, TVX has a 111 nucleotide deletion in its nonstructural (NS) gene, a 24 nucleotide insertion in VP1, and a 93 nucleotide repeat initiating from the C-terminus of the capsid gene., (Copyright © 2014 Vollmers et al.)

A set of proposals to rationalize and extend the taxonomy of the family Parvoviridae is currently under review by the International Committee on Taxonomy of Viruses (ICTV). Viruses in this family infect a wide range of hosts, as reflected by the longstanding division into two subfamilies: the Parvovirinae, which contains viruses that infect vertebrate hosts, and the Densovirinae, encompassing viruses that infect arthropod hosts. Using a modified definition for classification into the family that no longer demands isolation as long as the biological context is strong, but does require a near-complete DNA sequence, 134 new viruses and virus variants were identified. The proposals introduce new species and genera into both subfamilies, resolve one misclassified species, and improve taxonomic clarity by employing a series of systematic changes. These include identifying a precise level of sequence similarity required for viruses to belong to the same genus and decreasing the level of sequence similarity required for viruses to belong to the same species. These steps will facilitate recognition of the major phylogenetic branches within genera and eliminate the confusion caused by the near-identity of species and viruses. Changes to taxon nomenclature will establish numbered, non-Latinized binomial names for species, indicating genus affiliation and host range rather than recapitulating virus names. Also, affixes will be included in the names of genera to clarify subfamily affiliation and reduce the ambiguity that results from the vernacular use of "parvovirus" and "densovirus" to denote multiple taxon levels.

The 121-nucleotide left-end telomere of Minute Virus of Mice (MVM) can be folded into a Y-shaped hairpin with short axial ears that are highly conserved within genus Parvovirus. To explore their potential role(s) during infection, we constructed infectious plasmid clones that lacked one or other ear. Although these were nonviable when transfected into A9 cells, excision of the viral genome and DNA amplification appeared normal, and viral transcripts and proteins were expressed, but progeny virion production was minimal, supporting the idea of a potential role for the ears in genome packaging. To circumvent the absence of progeny that confounded further analysis of these mutants, plasmids were transfected into 293T cells both with and without an adenovirus helper construct, generating single bursts of progeny. These virions bound to A9 cells and were internalized but failed to initiate viral transcription, protein expression, or DNA replication. No defects in mutant virion stability or function could be detected in vitro. Significantly, mutant capsid gene expression and DNA replication could be rescued by coinfection with wild-type virions carrying a replication-competent, capsid-gene-replacement vector. To pinpoint where such complementation occurred, prior transfection of plasmids expressing only MVM nonstructural proteins was explored. NS1 alone, but not NS2, rescued transcription and protein expression from both P4 and P38 promoters, whereas NS1 molecules deleted for their C-terminal transactivation domain did not. These results suggest that the mutant virions reach the nucleus, uncoat, and are converted to duplex DNA but require an intact left-end hairpin structure to form the initiating transcription complex.

Engagement of innate viral sensors elicits a robust antiviral program via the induction of type I interferons (IFNs). Innate defense mechanisms against ssDNA viruses are not well defined. Here, we examine type I IFN induction and effectiveness in controlling a ssDNA virus. Using mouse embryonic fibroblasts (MEFs), we found that a murine parvovirus, minute virus of mice (MVMp), induced a delayed but significant IFN response. MEFs deficient in mitochondrial antiviral signaling protein (MAVS) mounted a wild-type IFN response to MVMp infection, indicating that RIG-I-dependent RNA intermediate recognition is not required for innate sensing of this virus. However, MVMp-induced IFNs, as well recombinant type I IFNs, were unable to inhibit viral replication. Finally, MVMp infected cells became unresponsive to Poly (I:C) stimulation. Together, these data suggest that the MVMp efficiently evades antiviral immune mechanisms imposed by type I IFNs, which may in part explain their efficient transmission between mice., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Toll-like receptor 9 (TLR9) recognizes genomes of double-stranded DNA (dsDNA) viruses in the endosome to stimulate plasmacytoid dendritic cells (pDCs). However, how and if viruses with single-stranded DNA (ssDNA) genomes are detected by pDCs remain unclear. Here we have shown that despite the ability of purified genomic DNA to stimulate TLR9 and despite the ability to enter TLR9 endosomes, ssDNA viruses of the Parvoviridae family failed to elicit an interferon (IFN) response in pDCs.

Parvoviruses have a linear single-stranded DNA genome, around 5 kb in length, with short imperfect terminal palindromes that fold back on themselves to form duplex hairpin telomeres. These contain most of the cis-acting information required for viral "rolling hairpin" DNA replication, an evolutionary adaptation of rolling-circle synthesis in which the hairpins create duplex replication origins, prime complementary strand synthesis, and act as hinges to reverse the direction of the unidirectional cellular fork. Genomes are packaged vectorially into small, rugged protein capsids ~260 Å in diameter, which mediate their delivery directly into the cell nucleus, where they await their host cell's entry into S phase under its own cell cycle control. Here we focus on genus-specific variations in genome structure and replication, and review host cell responses that modulate the nuclear environment.

Parvoviral terminal hairpins are essential for viral DNA amplification but are also implicated in multiple additional steps in the viral life cycle. The palindromes at the two ends of the minute virus of mice (MVM) genome are dissimilar and are processed by different resolution mechanisms that selectively direct encapsidation of predominantly negative-sense progeny genomes and conserve a single Flip sequence orientation at the 3' (left) end of such progeny. The sequence and predicted structure of these 3' hairpins are highly conserved within the genus Parvovirus, exemplified by the 121-nucleotide left-end sequence of MVM, which folds into a Y-shaped hairpin containing small internal palindromes that form the "ears" of the Y. To explore the potential role(s) of this hairpin in the viral life cycle, we constructed infectious clones with the ear sequences either inverted, to give the antiparallel Flop orientation, or with multiple transversions, conserving their base composition but changing their sequence. These were compared with a "bubble" mutant, designed to activate the normally silent origin in the inboard arm of the hairpin, thus potentially rendering symmetric the otherwise asymmetric junction resolution mechanism that drives maintenance of Flip. This mutant exhibited a major defect in viral duplex and single-strand DNA replication, characterized by the accumulation of covalently closed turnaround forms of the left end, and was rapidly supplanted by revertants that restored asymmetry. In contrast, both sequence and orientation changes in the hairpin ears were tolerated, suggesting that maintaining the Flip orientation of these structures is a consequence of, but not the reason for, asymmetric left-end processing.

The linear single-stranded DNA genome of minute virus of mice can be ejected, in a 3'-to-5' direction, via a cation-linked uncoating reaction that leaves the 5' end of the DNA firmly complexed with its otherwise intact protein capsid. Here we compare the phenotypes of four mutants, L172T, V40A, N149A, and N170A, which perturb the base of cylinders surrounding the icosahedral 5-fold axes of the virus, and show that these structures are strongly implicated in 3'-to-5' release. Although noninfectious at 37°C, all mutants were viable at 32°C, showed a temperature-sensitive cell entry defect, and, after proteolysis of externalized VP2 N termini, were unable to protect the VP1 domain, which is essential for bilayer penetration. Mutant virus yields from multiple-round infections were low and were characterized by the accumulation of virions containing subgenomic DNAs of specific sizes. In V40A, these derived exclusively from the 5' end of the genome, indicative of 3'-to-5' uncoating, while L172T, the most impaired mutant, had long subgenomic DNAs originating from both termini, suggesting additional packaging portal defects. Compared to the wild type, genome release in vitro following cation depletion was enhanced for all mutants, while only L172T released DNA, in both directions, without cation depletion following proteolysis at 37°C. Analysis of progeny from single-round infections showed that uncoating did not occur during virion assembly, release, or extraction. However, unlike the wild type, the V40A mutant extensively uncoated during cell entry, indicating that the V40-L172 interaction restrains an uncoating trigger mechanism within the endosomal compartment.

The parvovirus minute virus of mice (MVM) packages a single copy of its linear single-stranded DNA genome into preformed capsids, in a process that is probably driven by a virus-encoded helicase. Parvoviruses have a roughly cylindrically shaped pore that surrounds each of the 12 5-fold vertices. The pore, which penetrates the virion shell, is created by the juxtaposition of 10 antiparallel β-strands, two from each of the 5-fold-related capsid proteins. There is a bottleneck in the channel formed by the symmetry-related side chains of the leucines at position 172. We report here the X-ray crystal structure of the particles produced by a leucine-to-tryptophan mutation at position 172 and the analysis of its biochemical properties. The mutant capsid had its 5-fold channel blocked, and the particles were unable to package DNA, strongly suggesting that the 5-fold pore is the packaging portal for genome entry.

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We describe a structural rearrangement that can occur in parvovirus minute virus of mice (MVMp) virions following prolonged exposure to buffers containing 0.5 mM EDTA. Such particles remain stable at 4 degrees C but undergo a conformational shift upon heating to 37 degrees C at pH 7.2 that leads to the ejection of much of the viral genome in a 3'-to-5' direction, leaving the DNA tightly associated with the otherwise intact capsid. This rearrangement can be prevented by the addition of 1 mM CaCl(2) or MgCl(2) prior to incubation at 37 degrees C, suggesting that readily accessible divalent cation binding sites in the particle are critical for genome retention. Uncoating was not seen following the incubation of virions at pH 5.5 and 37 degrees C or at pH 7.2 and 37 degrees C in particles with subgenomic DNA, suggesting that pressure exerted by the full-length genome may influence this process. Uncoated genomes support complementary-strand synthesis by T7 DNA polymerase, but synthesis aborts upstream of the right-hand end, which remains capsid associated. We conclude that viral genomes are positioned so that their 3' termini and coding sequences can be released from intact particles at physiological temperatures by a limited conformational rearrangement. In the presence of divalent cations, incremental heating between 45 degrees C and 65 degrees C induces structural transitions that first lead to the extrusion of VP1 N termini, followed by genome exposure. However, in cation-depleted virions, the sequence of these shifts is blurred. Moreover, cation-depleted particles that have been induced to eject their genomes at 37 degrees C continue to sequester their VP1 N termini within the intact capsid, suggesting that these two extrusion events represent separable processes.

Background: Human bocavirus (HBoV) is a newly identified human parvovirus for which seroepidemiology and antigenic properties remain undefined., Methods: The HBoV VP2 gene, expressed from a baculovirus vector, produced virus-like particles (VLPs), which were used to raise rabbit anti-HBoV antisera and to develop an enzyme-linked immunosorbent assay (ELISA). The VLP-based ELISA was used to screen for HBoV-specific immunoglobulin G antibodies in a convenience sample of 270 serum specimens, mostly from children, obtained at Yale-New Haven Hospital; 208 specimens were also screened for erythrovirus B19-specific antibodies by a B19 VLP-based ELISA., Results: Immunofluorescence and ELISA showed that human parvoviruses HBoV and B19 are antigenically distinct. By the HBoV VLP-based ELISA, 91.8% and 63.6% of serum specimens from infants in the first and second months of life, respectively, were found to be seropositive, as were 45.4% from 3-month-old infants and 25.0% from 4-month-old infants. The percentages of HBoV-seropositive children increased to 40.7%-60.0% for children 5-47 months of age and to >85% for individuals >or=48 months old. However, the overall percentage of B19-seropositive individuals was <40.5% for all age groups screened., Conclusions: HBoV infection is common during childhood, but a minority of children and young adults screened have evidence of B19 infection.

To initiate DNA synthesis, the NS1 protein of minute virus of mice (MVM) first binds to a simple cognate recognition sequence in the viral origins, comprising two to three tandem copies of the tetranucleotide TGGT. However, this motif is also widely dispersed throughout the viral genome. Using an immunoselection procedure, we show that NS1 specifically binds to many internal sites, so that all viral fragments of more than approximately 170 nucleotides effectively compete for NS1, often binding with higher affinity to these internal sites than to sites in the origins. We explore the diversity of the internal sites using competitive binding and DNase I protection assays and show that they vary between two extreme forms. Simple sites with three somewhat degenerate, tandem TGGT reiterations bind effectively but are minimally responsive to ATP, while complex sites, containing multiple variably spaced TGGT elements arranged as opposing clusters, bind NS1 with an affinity that can be enhanced approximately 10-fold by ATP. Using immuno-selection procedures with randomized sequences embedded within specific regions of the genome, we explore possible binding configurations in these two types of site. We conclude that binding is modular, combinatorial, and highly flexible. NS1 recognizes two to six variably spaced, more-or-less degenerate forms of the 5'-TGGT-3' motif, so that it binds efficiently to a wide variety of sequences. Thus, despite complex coding constraints, binding sites are configured at frequent intervals throughout duplex forms of viral DNA, suggesting that NS1 may serve as a form of chromatin to protect and tailor the environment of replicating genomes.

Parvoviruses elaborate rugged nonenveloped icosahedral capsids of approximately 260 A in diameter that comprise just 60 copies of a common core structural polypeptide. While serving as exceptionally durable shells, capable of protecting the single-stranded DNA genome from environmental extremes, the capsid also undergoes sequential conformational changes that allow it to translocate the genome from its initial host cell nucleus all the way into the nucleus of its subsequent host. Lacking a duplex transcription template, the virus must then wait for its host to enter S-phase before it can initiate transcription and usurp the cell's synthetic pathways. Here we review cell entry mechanisms used by parvoviruses. We explore two apparently distinct modes of host cell specificity, first that used by Minute virus of mice, where subtle glycan-specific interactions between host receptors and residues surrounding twofold symmetry axes on the virion surface mediate differentiated cell type target specificity, while the second involves novel protein interactions with the canine transferrin receptor that allow a mutant of the feline leukopenia serotype, Canine parvovirus, to bind to and infect dog cells. We then discuss conformational shifts in the virion that accompany cell entry, causing exposure of a capsid-tethered phospholipase A2 enzymatic core that acts as an endosomolytic agent to mediate virion translocation across the lipid bilayer into the cell cytoplasm. Finally, we discuss virion delivery into the nucleus, and consider the nature of transcriptionally silent DNA species that, escaping detection by the cell, might allow unhampered progress into S-phase and hence unleash the parvoviral Trojan horse.

During DNA replication, the hairpin telomeres of Minute Virus of Mice (MVM) are extended and copied to create imperfectly palindromic duplex junction sequences that bridge adjacent genomes in concatameric replicative-form DNA. These are resolved by the viral initiator protein, NS1, but mechanisms employed at the two telomeres differ. Left-end:left-end junctions are resolved asymmetrically at a single site, OriLTC, by NS1 acting in concert with a host factor, parvovirus initiation factor (PIF). Replication segregates doublet and triplet sequences, initially present as unpaired nucleotides in the bubble region of the left-end hairpin stem, to either side of the junction. These act as spacers between the NS1 and PIF binding sites, and their asymmetric distribution sets up active (OriLTC) and inactive (OriLGAA) forms of OriL. We used a reverse genetic approach to disrupt this asymmetry and found that neither opposing doublets nor triplets in the hairpin bubble were tolerated. Viable mutants were isolated at low frequency and found to contain second-site mutations that either restored the asymmetry or crippled one PIF binding site. These mutations either inactivated the inboard or activated the outboard form of OriL, a polarity that strongly suggests that, in the genus Parvovirus, an active inboard OriL is lethal.

Cylindrical projections surrounding the fivefold-symmetry axes in minute virus of mice (MVM) harbor central pores that penetrate through the virion shell. In newly released DNA-containing particles, these pores contain residues 28 to 38 belonging to a single copy of VP2, disposed so that its extreme N-terminal domain projects outside the particle. Virions are metastable, initially sequestering internally the N termini of all copies of the minor capsid protein, VP1, that is essential for entry. This VP1 domain can be externalized in vitro in response to limited heating, and we show here that the efficiency of this transition is greatly enhanced by proteolysis of VP2 N termini to yield VP3. This step also renders the VP1 rearrangement pH dependent, indicating that VP2 cleavage is a maturation step required to prime subsequent emergence of the VP1 "entry" domain. The tightest constriction within the cylinder is created by VP2 leucine 172, the five symmetry-related copies of which form a portal that resembles an iris diaphragm across the base of the pore. In MVMp, threonine substitution at this position, L172T, yields infectious particles following transfection at 37 degrees C, but these can initiate infection only at 32 degrees C, and this process can be blocked by exposing virions to a cellular factor(s) at 37 degrees C during the first 8 h after entry. At 32 degrees C, the mutant particle is highly infectious, and it remains stable prior to VP2 cleavage or following cleavage at pH 5.5 or below. However, upon exposure to neutral pH following VP2 cleavage, its VP1-specific sequences and genome are extruded even at room temperature, underscoring the significance of the VP2 cleavage step for MVM particle dynamics.

Two host range switch mutants of the immunosuppressive strain of parvovirus Minute Virus of Mice (MVMi) were isolated from plaques on A9 fibroblasts. Both carried a single coding mutation at residue D399 in VP2, to alanine and glycine in hr105 and hr107, respectively, and a second, non-coding, guanine-to-adenine change at nucleotide 1970 in hr105 and 1967 in hr107. These mutations were recreated in a wild type MVMi infectious plasmid clone, both alone and as pairs, in either the original or switched combinations. All single mutants failed to replicate productively in fibroblasts, but the two pairs of changes were functionally equivalent. Single D399 mutations allowed the viruses to initiate infection in fibroblasts, but NS2 expression was severely restricted and correlated with poor accumulation and release of progeny virus. Mutations at 1967 or 1970 enhanced NS2 accumulation, and allowed efficient progeny production and release. Conversely, the D399 mutations destroyed the viruses' ability to infect EL4 lymphocytes. In all productive EL4 infections, NS2 was expressed at high ratios even in the absence of upstream mutations, and progeny accumulation was efficient. However, EL4 cells lack a mechanism for early progeny release, potentially explaining why virus amplification in these cells is slow.

Minute virus of mice (MVM) packages a single, negative-sense copy of its linear single-stranded DNA genome, but a chimeric virus, MML, in which >95% MVM sequence was fused to the right-hand terminus of LuIII, packages >40% positive-sense DNA. While encapsidation of both MML strands begins efficiently, genome translocation frequently stalls at specific sites in positive-sense DNA. Internalized sequences, derived from the 3' end of the strand, ranged from 1 to 5 kb in length, with species of around 2 kb predominating. When nuclease activity during isolation was minimized, these truncated species were found to be part of pre-excised 5 kb single-strands. Similarly, some partially encapsidated negative-sense DNAs were observed, forming a continuum of protected 3' sequences between 1 and 3 kb in length, but these were less abundant and more uniformly distributed than their positive-sense counterparts, indicating that the negative strand has evolved for efficient internalization. The paucity of protected DNAs shorter than 1-2 kb suggests that translocation is biphasic, proceeding efficiently through the first (3') third of the genome, but prone to stall thereafter. Sequences with conspicuous secondary structure, including stem-loop and guanidine rich regions, were found to interrupt packaging, especially when positioned near the 5' end of the strand. Since VP2 amino-terminal peptides were exposed at the particle surface in all packaging intermediates, extrusion of this peptide precedes translocation of the full-length strand.

The parvovirus minute virus of mice (MVM) packages predominantly negative-sense single strands, while its close relative LuIII encapsidates strands of both polarities with equal efficiency. Using genomic chimeras and mutagenesis, we show that the ability to package positive strands maps not, as originally postulated, to divergent untranslated regions downstream of the capsid gene but to the viral hairpins and predominantly to the nick site of OriR, the right-end replication origin. In MVM, the sequence of this site is 5'-CTAT(black triangle down)TCA-3', while in LuIII a two-base insertion (underlined) changes it to 5'-CTATAT(black triangle down)TCA-3'. Matched LuIII genomes differing only at this position (designated LuIII and LuDelta2) packaged 47 and <8% positive-sense strands, respectively. OriR sequences from these viruses were both able to support NS1-mediated nicking in vitro, but initiation efficiency was consistently two- to threefold higher for LuDelta2 derivatives, suggesting that LuIII's ability to package positive strands is determined by a suboptimal right-end origin rather than by strand-specific packaging sequences. These observations support a mathematical "kinetic hairpin transfer" model, previously described by Chen and colleagues (K. C. Chen, J. J. Tyson, M. Lederman, E. R. Stout, and R. C. Bates, J. Mol. Biol. 208:283-296, 1989), that postulates that preferential excision of particular strands is solely responsible for packaging specificity. By analyzing replicative-form (RF) DNA generated in vivo during LuIII and LuDelta2 infections, we extend this model, showing that positive-sense strands do accumulate in LuDelta2 infections as part of duplex RF DNA, but these do not support packaging. However, replication is biphasic, so that accumulation of positive-sense strands is ultimately suppressed, probably because the onset of packaging removes newly displaced single strands from the replicating pool.

The minute virus of mice initiator protein, NS1, excises new copies of the left viral telomere in a single sequence orientation, dubbed flip, during resolution of the junction between monomer genomes in palindromic dimer intermediate duplexes. We examined this reaction in vitro using both (32)P-end-labeled linear substrates and similar unlabeled templates labeled by incorporation of [alpha-(32)P]TTP during the synthesis. The observed products suggest a resolution model that explains conservation of the hairpin sequence and in which a novel heterocruciform intermediate plays a crucial role. In vitro, NS1 initiates two replication pathways from OriL(TC), the single active origin embedded in one arm of the dimer junction. NS1-mediated nicking liberates a base-paired 3' nucleotide to prime DNA synthesis and, in a reaction we call "read-through synthesis," forks established while the substrate is a linear duplex synthesize DNA in the flop orientation, leading to DNA amplification but not to junction resolution. Nicking leaves NS1 covalently attached to the 5' end of the DNA, where it can serve as a 3'-to-5' helicase, unwinding the NS1-associated strand. In the second pathway, resolution substrates are created when such unwinding induces the palindrome to reconfigure into a cruciform prior to fork assembly. New forks can then synthesize DNA in the flip orientation, copying one cruciform arm and creating a heterocruciform intermediate. Resolution proceeds via hairpin transfer in the extended arm of the heterocruciform, which releases one covalently closed duplex telomere and a partially single-stranded junction intermediate. We suggest that the latter intermediate is finally resolved via an NS1-induced single-strand nick at the otherwise inactive origin, OriL(GAA).

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., (Copyright 2001 Academic Press.)

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.

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.

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.

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.

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., (Copyright 1999 Academic Press.)

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

Regions of open reading frame (ORF) from the genome of the autonomous parvovirus minute virus of mice (MVM) were cloned into a procaryotic expression vector, and bacterial fusion proteins containing MVM-specific amino acid sequences were isolated. Antibodies raised against these proteins were used to immunoprecipitate viral proteins synthesized in vitro in a rabbit reticulocyte lysate translation system programmed with mRNA isolated from cells infected with MVM and a number of different parvoviruses. These studies demonstrated that: the 83-kilodalton nonstructural protein NS-1 and the 25-kilodalton nonstructural protein NS-2 have a common amino-terminal sequence which is encoded by the single ORF located between nucleotides 225 and 534 in the viral genome; the ORF located between nucleotides 1110 and 1638 is only expressed in the NS-1 protein; and the sequence encoded in a small alternative ORF between nucleotides 2075 and 2291 is expressed exclusively in NS-2. These data confirm that NS-1 is the product of the 4.8-kilobase R1 viral transcript and demonstrate that NS-2 is synthesized from the 3.3-kilobase R2 transcript which arises from the left-hand promoter at map unit 4 on the viral genome. Antibodies against the MVM fusion proteins also cross-reacted with similar proteins encoded by the viruses H-1 and LuIII, but although antibodies against the carboxy-terminal half of NS-1 cross-reacted with a similar protein in CPV, we were unable to demonstrate an NS-2 protein encoded by this virus.

The nucleotide sequence of an almost-full-length clone of human parvovirus B19 was determined. Whereas the extreme left and right ends of this genomic clone are incomplete, the sequence clearly indicates that the two ends of viral DNA are related by inverted terminal repeats similar to those of the Dependovirus genus. The coding regions are complete in the cloned DNA, and the two large open reading frames which span almost the entire genome are restricted to one strand, as has been found for all other parvoviruses characterized to date. From the DNA sequence we conclude that the organization of the B19 transcription units is similar although not identical to those of other parvoviruses. In particular, we predict that the B19 genome may utilize a fourth promoter to transcribe mRNA encoding the major structural polypeptide, VP2. Analysis of the putative polypeptides confirms that B19 is only distantly related to the other parvoviruses but reveals that there is a small region in the gene probably encoding the major nonstructural protein of B19, which is closely conserved between all of the parvovirus genomes for which sequence information is currently available.