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We have studied the human thymus microenvironment in order to identify subsets of cells that may be responsible for the induction of different aspects of T-lymphocyte differentiation, education and MHC restriction. Using immunofluorescence on tissue sections and cell suspensions we have found MHC products (HLA-A, B, C and DR) to be present throughout the thymus epithelium whilst human T-cell antigens are absent from all non-lymphoid cells. In contrast, Thy-1 antigen (expressed on ∼ 1% paediatric human thymocytes) has a differential expression amongst thymic epithelial cells, being confined to those in the subcapsular cortex and to ‘thymic nurse cells’ (TNC). The former represent the site to which thymocyte precursors first migrate upon entering the thymus. The latter ate large epithelial cells, located within the cortex, whose plasma membrane totally encloses a number of thymus lymphocytes; these cells are therefore good candidates for the mediators of direct contact (stromal) induced thymocyte maturation. [ABSTRACT FROM AUTHOR]

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.

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.

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 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.

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.

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.

Cells infected with the autonomous parvovirus MVM synthesize a major virally coded non-structural protein which accumulates in the nucleus and is phosphorylated to a significant extent. Peptide map analysis shows that this in vivo product has the same primary sequence as the NS-1 protein previously identified in the in vitro translation products of MVM encoded mRNA, and as such is the product of the largest messenger RNA species, a spliced 4.8 kb (R1) transcript. In vivo NS-1 exists in two predominant forms, a phosphorylated 84-85 kDa species and an unphosphorylated or poorly phosphorylated 83 kDa form. Both forms are preferentially extracted from the nucleus using an extraction procedure which enriches for replication complexes.

The genome of the small human virus serologically associated with erythrocyte aplasia and erythema infectiosum (fifth disease) is shown to be a linear, nonpermuted, single-stranded DNA molecule with self-priming hairpin termini, properties which are characteristic of the genomes of the family Parvoviridae. This human parvovirus chromosome was molecularly cloned into bacterial plasmid vectors and the cloned DNA was used to explore its relatedness to other mammalian parvovirus serotypes by DNA:DNA hybridization. It is not related to the human adeno-associated viruses but does show a distant evolutionary relationship to genomes of the helper-independent parvoviruses of rodents. This strongly suggests that it is an autonomous parvovirus, and as such is the first example of a member of this group of common animal pathogens to cause disease in man.

The Thy-1 antigen is a cell surface glycoprotein found in neural tissue of all mammalian species so far studied. The distribution and amount of this antigen has been measured on 4 human neuronal and 2 neuroglial cell lines and on fresh tumour cells of neuronal origin. In 3 out of 4 neuronal lines (LAN-1, TR14, CHP 212) more than 90% of cells were Thy-1+, however, LAN-1 cells showed only weak immunofluorescence and bore on average 2.4 times fewer molecules of Thy-1 per cell than those of either TR14 or CHP 212. The mean number of Thy-1 molecules per TR14 cell was shown to be approximately 2.25 x 10(5). In contrast, only 66% of cells in the fourth neuronal line (CHP 100) were Thy-1+, although these showed strong immunofluorescence. Both glial cell lines, UCH-203 and H314/123, showed strong Thy-1 immunofluorescence on more than 90% of cells. Similarly, with fresh neuronal tumour cells, although approximately 80% of tumours were Thy-1+ (essentially 100% of cells in these being positive) there were considerable differences in the intensity of labelling by immunofluorescence between different tumours. Such heterogeneity in cell lines and malignancy may reflect normal in vivo variation. Different phenotypes might therefore represent separate neural cell lineages, or simply differences in maturational status within a lineage. The very low frequency of Thy-1+ cells in normal bone marrow (less than 0.1% of nucleated cells) indicates that anti-Thy-1 antibodies may be valuable in both the diagnosis and subsequent treatment of neuroblastoma.

In vitro translation of mRNA from cells infected with the autonomous parvovirus MVM yields four major virally coded proteins. Two of these proteins are indistinguishable both antigenically and by peptide map analysis from the viral capsid polypeptides VP-1 and VP-2. The other two proteins, designated NS-1 and NS-2, are not related to the capsid polypeptides but are recognized by sera from animals infected with different autonomous parvovirus serotypes. The NS-1 protein made in vitro comigrates with VP-1 (MW approximately 83,000), while the NS-2 polypeptide has an apparent molecular weight of 24,000. The transcript for the NS-1 polypeptide was mapped to a block of open reading frame located in the major intron of the left-hand transcription unit in the MVM genome.

We have studied the human thymus microenvironment in order to identify subsets of cells that may be responsible for the induction of different aspects of T-lymphocyte differentiation, education and MHC restriction. Using immunofluorescence on tissue sections and cell suspensions we have found MHC products (HLA-A, B, C and DR) to be present throughout the thymus epithelium whilst human T-cell antigens are absent from all non-lymphoid cells. In contrast, Thy-1 antigen (expressed on approximately 1% paediatric human thymocytes) has a differential expression amongst thymic epithelial cells, being confined to those in the subcapsular cortex and to 'thymic nurse cells' (TNC). The former represent the site to which thymocyte precursors first migrate upon entering the thymus. The latter are large epithelial cells, located within the cortex, whose plasma membrane totally enclose a number of thymus lymphocytes; these cells are therefore good candidates for the mediators of direct contact (stromal) induced thymocyte maturation.

The FR3T3 and NRK rat cell lines and their human EJ Ha-ras-1 oncogene-transformed derivatives, termed FREJ and NREJ, were compared for their susceptibility to the parvovirus MVMp. For a similar production of p21ras protein, FREJ clones are markedly sensitized to killing by MVMp, whereas the NREJ cells are not. Such a contrasting effect of ras transformation on the sensitivity of cells of different origins to MVMp can be traced back to their respective abilities to support the parvoviral life cycle. The FR3T3 line produces a substantial amount of viral DNA whose expression in the form of the nonstructural protein NS-1 is stimulated in its transformed derivatives. Conversely, NRK cells offer an early block to parvoviral DNA replication and expression that appears to persist in the ras-transformed clones. Thus, at least two intracellular restrictions can protect normal rat cells against MVMp infection, and transformation by ras relieves one of them at the level of parvoviral gene expression. A fair correlation was also found between the degree of sensitivity of the various lines to MVMp-induced killing and their capacity to synthesize the nonstructural viral proteins, suggesting a possible role of parvoviral nonstructural proteins in cytotoxicity.