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The small 11-kDa proteins of B19 parvovirus contain three proline-rich regions which conform to consensus Src homology 3 (SH3) ligand sequences present in signaling molecules within the cell. We have shown that the B19 11-kDa proteins specifically interact with the growth factor receptor-binding protein 2 (Grb2) in vitro. Mutation of prolines within one of the three SH3 ligand-like sequences decreases the binding of B19 11-kDa proteins to Grb2, suggesting that the proline-rich region is involved in the B19 11-kDa/Grb2 interaction. Therefore, the B19 11-kDa proteins may function to alter Grb2-mediated signaling by disrupting SH3 domain/ligand interactions. These results implicate the 11-kDa proteins in B19 pathogenesis through perturbation of normal cellular signaling pathways., ((C)2001 Elsevier Science.)

NS1, the major nonstructural parvovirus protein of the minute virus of mice, is a multifunctional protein responsible for several aspects of viral replication. NS1 transactivates the P38 promoter (used to express the structural proteins), as well as its own strong promoter, P4. To study the mechanism of activation and to map regions of NS1 responsible for transactivation, NS1 and various deletions of NS1 were cloned in frame with the GAL4DB and cotransfected into COS-7 and LA9 cells with a synthetic GAL4-responsive reporter plasmid. These studies showed NS1 can directly activate transcription through its 129 carboxyl-terminal amino acid residues. Any deletion from this region of the C terminus, even as few as 8 amino acids, completely abolishes transactivation. A yeast two-hybrid system used to identify protein-protein interactions demonstrated that NS1 is able to dimerize when expressed in yeast cells. However, only an almost complete NS11-638 bait was able to interact with the full-length NS1. A two-hybrid screen identified a HeLa cell cDNA clone (NS1-associated protein 1 [NSAP1]) that interacts with NS11-276 and NS11-638. An additional sequence was predicted from human EST (expressed sequence tag) data, and the cDNA was estimated to be at least 2,221 bp long, potentially encoding a 562-amino-acid protein product. A polyclonal antibody raised to a synthetic peptide within NSAP1 recognizes an approximately 65-kDa cellular protein. This NSAP1 cDNA has not previously been characterized, but the predicted protein sequence is 80% identical to the recently identified heterogeneous nuclear ribonucleoprotein (hnRNP) R (W. Hassfeld et al., Nucleic Acids Res. 26:439-445, 1998). NSAP1 contains four ribonucleoprotein domains, as well as a highly repetitive C-terminal region. A closely related mouse cDNA (deduced from murine EST data) encodes a protein with only a single amino acid residue change from the human protein. NSAP1 is predicted to be a 65-kDa polynucleotide binding protein, and it likely functions in the regulation of splicing and/or transport of mRNAs from the nucleus.

During replication of the minute virus of mice (MVM) genome, a dimer replicative form (RF) intermediate is resolved into two monomer RF molecules in such a way as to retain a unique sequence within the left hand hairpin terminus of the viral genome. Although the proposed mechanism for resolution of the dimer RF remains uncertain, it likely involves site-specific nicking of the dimer bridge. The RF contains two double-stranded copies of the viral genome joined by the extended 3' hairpin. Minor sequence asymmetries within the 3' hairpin allow the two halves of the dimer bridge to be distinguished. The A half contains the sequence [sequence: see text], whereas the B half contains the sequence [sequence: see text]. Using an in vitro assay, we show that only the B half of the MVM dimer bridge is nicked site-specifically when incubated with crude NS-1 protein (expressed in insect cells) and mouse LA9 cellular extract. When highly purified NS-1, the major nonstructural protein of MVM, is used in this nicking reaction, there is an absolute requirement for the LA9 cellular extract, suggesting a cellular factor (or factors) is (are) required. A series of mutations were created in the putative host factor binding region (HFBR) on the B half of the MVM dimer bridge adjacent to the NS-1 binding site. Nicking assays of these B half mutants showed that two CG motifs displaced by 10 nucleotides are important for nicking. Gel mobility shift assays demonstrated that a host factor(s) can bind to the HFBR of the B half of the dimer bridge and efficient binding depends on the presence of both CG motifs. Competitor DNA containing the wild-type HFBR sequence is able to specifically inhibit nicking of the B half, indicating that the host factor(s) bound to the HFBR is(are) essential for site-specific nicking to occur.

The minute virus of mice (MVM) genome is a linear single-stranded length of approximately 5000 nucleotides of DNA with unique terminal palindromic sequences at both ends. The left(3') hairpin is used to prime the initiation of DNA synthesis on parental single-strand DNA while the right (5') hairpin or stem-plus-arms structure can also prime the initiation of DNA synthesis during synthesis of dimer and higher oligomers as well as synthesis of progeny single strands. Previous studies have shown that if viral duplex DNA was input into an in vitro DNA replication system using extracts from uninfected HeLa cells, the 5' end of the molecule was able to form a hairpin and initiate DNA synthesis by DNA polymerase delta (Cossons et al. (1996), Virology 216, 258-264). In this study, the effect of the deletion of known cis-acting genetic elements upon the initiation of DNA replication was studied using a series of MVM mutants with deletions within the 5' terminal region. Mutants containing deletions of elements A (nucleotides 4489-4636), B (nucleotides 4636-4695), and either one or both of the 65-bp repeats (nucleotides 4720-4785 and 4785-4849) were used as template in the in vitro DNA replication system. When element A was deleted, the efficiency of initiation decreased significantly. Subsequent removal of element B, leaving just the two 65-bp repeats, restored levels of initiation back to those seen in the wild-type genome. In the absence of either A or B both 65-bp repeats were necessary for efficient initiation, and removal of one of these repeats caused a decrease in efficiency. Thus, element B appeared to have a negative regulatory effect (in the absence of element A), and element A appeared to have a positive regulatory effect, at least in the presence of element B. These data demonstrate, for the first time, a complex interaction between these cis-acting regulatory elements which can function as both positive or negative regulators in the initiation of MVM DNA replication.

Previous characterization of the terminal sequences of the minute virus of mice (MVM) genome demonstrated that the right hand palindrome contains two sequences, each the inverted complement of the other. However, the left hand palindrome was shown to exist as a unique sequence [Astell et al., J. Virol. 54: 179-185 (1985)]. The modified rolling hairpin (MRH) model for MVM replication provided an explanation of how the right hand palindrome could undergo hairpin transfer to generate two sequences, while the left end palindrome within the dimer bridge could undergo asymmetric resolution and retain the unique left end sequence. This report describes in vitro resolution of the wild-type dimer bridge sequence of MVM using recombinant (baculovirus) expressed NS-1 and a replication extract from LA9 cells. The resolution products are consistent with those predicted by the MRH model, providing support for this replication mechanism. In addition, mutant dimer bridge clones were constructed and used in the resolution assay. The mutant structures included removal of the asymmetry in the hairpin stem, inversion of the sequence at the initiating nick site, and a 2-bp deletion within one stem of the dimer bridge. In all cases, the mutant dimer bridge structures are resolved; however, the resolution pattern observed with the mutant dimer bridge compared with the wild-type dimer bridge is shifted toward symmetrical resolution. These results suggest that sequences within the left hand hairpin (and hence dimer bridge sequence) are responsible for asymmetric resolution and conservation of the unique sequence within the left hand palindrome of the MVM genome.

The NS-1 protein of minute virus of mice (MVM) is required for viral DNA replication and transcriptional regulation. To define the domain structure of NS-1, we have generated point mutations in its putative NTP-binding/ATPase domain. We show that all mutants were unable to support replication of MVM DNA in a transient DNA replication assay. Furthermore, all mutants, except for the K405S substitution, were able to transactivate the P38 promoter in transient transfection experiments. NS-1 proteins bearing COOH-terminal deletions of 29 and 33 amino acid residues were also transcriptionally inert. Biochemical analysis of recombinant NS-1 expressed in insect cells shows that mutations in the putative NTP-binding/ATPase domain severely reduced helicase activity in vitro. However, affinity labeling experiments indicate that none of these mutations, except for K469T, impaired NTP-binding activity. Finally, all point mutants retained significant levels of ATPase activity, except for the E444Q mutant (1%). These findings suggest that the replication and transcription activities of NS-1 reside in separate functional domains. In addition, NS-1 proteins with mutations in the putative nucleotide binding fold have lost helicase activity, whereas most retain nucleotide binding and ATPase functions, suggesting that the mutations have uncoupled the ATPase and helicase activities.

A second small ORF contained within the parvovirus B19 genome has been shown to direct synthesis of a small 7.5-kDa protein. These results extend our previous studies in which we identified a family of 11-kDa proteins (J. St. Amand, C. Beard, K. Humphries, and C.R. Astell, Virology 183, 133-142, 1991; J. St. Amand and C.R. Astell, Virology, 192, 121-131, 1993). The presumed ORF encoding the 7.5-kDa protein was discovered on the left side of the viral genome and was identified by expression of the protein and in vitro mutagenesis. Two small RNA transcripts of B19 virus were demonstrated to be able to direct the synthesis of this 7.5-kDa protein by in vitro translation using rabbit reticulocyte lysates. Expression of 7.5-kDa protein was also observed in COS-7 cells transfected with a plasmid containing the B19 genome as well as in human peripheral mononucleocytes infected with B19 virus. In both cases the 7.5-kDa protein was detected by immunoprecipitation of cell lysates with polyclonal antibodies raised against a synthetic peptide corresponding to a portion of the predicted protein sequence. These data partially reveal the function of the abundant small RNA transcripts produced by parvovirus B19.

In this report, we describe the replication of minigenomes of minute virus of mice (MVM). We show that the cis-acting sequences required for MVM DNA replication reside in the terminal 140 and 660 nucleotides of the left and right termini, respectively. Minigenomes containing either two right (RR) or two left (LL) termini are replication competent genomes, demonstrating that both termini contain the genetic information necessary for the excision and initiation of DNA replication. Since the efficiency of replication of the RR genome is greater than that of the LL genome, it suggests that the individual terminal sequences are not equivalent in function. In addition to the terminal palindromic sequences required for replication, we show that specific elements found inboard of the right hairpin between nucleotides 4489-4636 (element A) and 4636-4695 (element B) are necessary for the efficient replication of MVM minigenomes. These elements have heretofore not been identified as replication sequences.

The human pathogenic parvovirus B19 directs the synthesis of two size classes of small abundant RNAs. It is shown that the smallest RNAs, of 500 and 600 nt, are translated into at least two 11-kDa proteins in B19-infected human leukemic bone marrow cells. A COS-7 cell expression system was used to demonstrate that the different forms of the protein result from translational initiation at multiple AUG codons in the same 94 aa ORF. The 11-kDa proteins were localized to the cytoplasm of transfected COS-7 cells using indirect immunofluorescence. However, their localization was at least partially nuclear in B19-infected cells. In COS-7 cells the expression of the major B19 structural and nonstructural proteins was not affected in the absence of the expression of the 11-kDa proteins.

The gene encoding the major nonstructural (NS-1) protein of minute virus of mice (MVM) has been expressed in insect cells using a baculovirus expression system. This 83-kDa polypeptide was found to be localized in the soluble (cytosolic) fraction in insect cells, in contrast with the nuclear localization of NS-1 expressed in MVM-infected mouse LA-9 cells. The protein was purified by immunoaffinity chromatography using a monoclonal antibody (MAb) prepared to an NS-1 fusion peptide [(Yeung et al., Virology 185, 35-45 (1991)]. Recombinant NS-1 was eluted using either low pH or a synthetic peptide corresponding to the epitope of the MAb. The peptide-eluted material is greater than 95% pure and biologically active in that it has ATPase activity and ATP-dependent helicase activity as determined by a strand displacement assay.

Two parvovirus B19 cDNA libraries have been constructed; one from COS-7 cells transfected with a B19/pSVOd hybrid vector and the other from B19-infected human erythroid leukemic cells. We have used these libraries to investigate the expression of the abundant classes of polyadenylated B19 RNAs; the 700- and 800-nt class which terminates in the middle of the genome and the 500- and 600-nt class which contains an ORF from the extreme right-hand end of the genome. The 700- and 800-nt RNA species were not found in the COS cell library, suggesting that a variant polyadenylation signal (ATTAAA or AATAAC) in the middle of the genome is not efficiently recognized in these cells. In contrast, the 700- and 800-nt class was highly represented in the human library, confirming the use of this variant polyadenylation signal in the normal host cell of the virus. In COS cells the middle exon of the 500- and 600-nt class of RNA exhibited variability in both splice donor and acceptor sites. However, in human cells there were only two splice acceptor sites nt 1910 and 2030, and a single splice donor site nt 2183 for this exon. Antisera, prepared against a peptide derived from the 94-aa potential protein encoded by the 500- and 600-nt class of RNA, recognized, on a Western blot, a polypeptide of approximately 11 kDa that was translated in vitro from these cDNAs and in vivo in pSVOd/B19 transfected COS cells. Immunoprecipitation revealed that two proteins were made from this ORF, suggesting the use of internal translation initiation site(s). Another antisera, raised against a second peptide corresponding to an antigenic region of the potential protein encoded by the 700- and 800-nt class of RNA, failed to detect a 15-kDa protein by Western blotting or immunoprecipitation of labeled proteins both in vitro and in vivo in COS cells.

Monoclonal antibodies were raised against a bacterial fusion protein containing amino acids 364 to 623 of the major nonstructural protein, NS-1, of minute virus of mice (MVMp), an autonomous parvovirus. By immunoblot analyses, these antibodies all recognized an 83-kDa protein in MVM-infected mouse fibroblast cells. Indirect immunofluorescence studies showed that five of the six react against a nuclear protein in MVM-infected mouse cells resulting in discrete foci of fluorescence. These foci do not correspond with the nucleoli, the site of MVM DNA replication. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen and showed that four distinct epitopes were recognized by the different antibodies. A 25-amino-acid peptide was used in competition ELISAs to confirm the location of the epitope recognized by two antibodies CE10 and AC6. Preliminary characterization of an NS-1/NS-2 fusion protein synthesized in insect cells using a baculovirus expression vector showed that this fusion protein is also localized within the nucleus; however, in contrast, the full-length NS-1 polypeptide is located within the cytoplasm.

Segments of cloned Drosophila DNA from four recombinant plasmids that hybridize with tRNA4Val have been sequenced. The segments from pDt92R and pDt120R that hybridize to 90C on the third polytene chromosome appear to be either repeats or alleles. They contain one structural gene each of identical sequence but differ at eight sites in 506 base pairs. The structural genes differ at four sites from the sequence expected from that of tRNA4Val. A third plasmid, pDt14, which hybridizes to 89BC on the third chromosome, also contains a structural gene with the same sequence as those in pDt92R and pDt120R. In addition, pDt14 has a gene for tRNA2Phe 214 base pairs upstream and with the same polarity as the tRNA4Val gene. The tRNA2Phe gene contains a 23-base pair segment identical with the corresponding segment in the tRNA4Val genes except for one base pair. The fourth plasmid investigated, pDt55, hybridizes to 70BC. It contains two tRNA4Val genes 525 base pairs apart with opposite polarity. These genes have identical sequences, which corresponds to that expected from the sequence of tRNA4Val. There is no evidence that the first three tRNA4Val genes are expressed at any stage during the development of Drosophila.

Hybrid B19 parvovirus-SV40 origin vectors were transfected into COS-7 cells and replication of these plasmids studied. Plasmids that have a frameshift mutation within the nonstructural gene region replicated to high level (copy number approximately 10,000/transfected cell) although somewhat lower than pSVOd, the SV40 origin vector without B19 sequence (copy number approximately 100,000/transfected cell). However, hybrid B19 parvovirus-SV40 origin vectors that do not contain these frameshift mutations replicated to a much lower level (copy number approximately 1000/transfected cell). Although the hybrid vectors studied replicated at different efficiencies in COS-7 cells, they are transcribed at approximately the same level, resulting in RNA species that are indistinguishable from those seen in B19 virus-infected erythroid bone marrow cells. Western blot analysis demonstrated that the mRNAs are translated into polypeptides of the same size and, in the case of viral structural proteins, in same relative abundance as seen in a B19-infected clinical sample.

We have determined the complete nucleotide sequence of the genome of Minute Virus of Mice, an autonomous parvovirus. This single-stranded DNA is 5081 nucleotides long. The 3'-and 5'-ends of the viral strand contain imperfect palindromic sequences which consist, respectively, of 115 and 206 nucleotides. The 3'-terminal palindrome is composed of a unique sequence, whereas the 5'-terminal palindrome contains two sequences in equimolar amounts; these are related in that one is the inverted complement of the other. The DNA strand complementary to that which is encapsidated into virions contains two large open reading frames which together span almost the entire genome. Transcriptional and translational signals within the sequence have been identified and related to the known map coordinates of the viral transcripts. In this report we summarize some of the salient structural and organizational features of the MVM genome.

The deletion events that have plagued attempts to maintain molecular clones with long palindromic DNA sequences in Escherichia coli have been shown to be less frequent in recBCsbcB hosts [Collins et al., Gene 19 (1982) 139-146]. This study sought to determine if such hosts would permit the stable propagation of plasmid clones carrying the deletion-generating, 206 nucleotide (nt) long, imperfect palindrome derived from the 5' terminus of the genome of minute virus of mice (MVM), an autonomous parvovirus [Astell et al., Nucleic Acids Res. 11 (1983) 999-1018]. To this end these hybrid plasmids were used to transform several different mutant recBCsbcB hosts, followed by the isolation and restriction mapping of plasmid DNA from selected transformants. Characterization of plasmid DNA isolated from a recBCsbcBrecF host indicated deletion-resistant propagation of the intact species. Sequence analysis of unamplified and chloramphenicol (Cm)-amplified plasmid DNA obtained from these clones confirmed the integrity of the palindromic region of the viral DNA insert.

The nucleotide sequence of the B19-Wi isolate of human parvovirus was determined and compared throughout the open reading frames and putative transcription signals with the sequence of the closely related B19-Au isolate. In vitro run off transcription assays, using B19-Wi DNA as the template, indicated that there is a strong promoter between m.u. 5 and 7. Deletion clones show that a region between nt 258 and 321 is necessary for in vitro transcriptional activity. Primer extension studies identified the start site at 31-32 nucleotides downstream of the sequence TATATATA. The strength of this left-hand promoter is unusual among parvovirus promoters characterized to date, and the possibility of an upstream enhancer element is discussed.

A decadeoxyribonucleotide, pAAATCCCTCA, was synthesized to complement nucleotides 2920--2930 of coliphage phi X174 viral DNA except that the nucleotide corresponding to position 2925 is deleted. The phage DNA sequence in this region codes for the ribosome-binding site of gene H. The oligodeoxyribonucleotide was integrated into double-stranded phi X174 DNA by using it as a primer for DNA polymerase with wild-type DNA template followed by ligation. The resultant heteroduplex DNA was used to transfect Escherichia coli spheroplasts and progeny bacteriophage were isolated. The synthetic decadeoxyribonucleotide was then used to enrich mutant viral DNA from which nucleotide 2925 had been deleted. After three cycles of enrichment and spheroplast transfection only mutant DNA was detectable. The deletion of nucleotide 2925 from the phi X174 genome seriously disrupts a sequence complementary to the 3'-terminus of the 16S rRNA of E. coli and also eliminates a translation termination codon from that sequence. However, the mutant grows normally with no readily perceptible phenotype. Thus, a short synthetic oligodeoxyribonucleotide has been used to construct, and to isolate with 100% efficiency, a mutant for which there is no biological selection procedure.

We have developed a standardized, quantitative assay to study the function of a cloned adenovirus origin. We have shown that the adenovirus origin is located within the first 20 bp of the adenovirus inverted terminal repetition (ITR), a region containing a sequence conserved among human, simian, murine, and avian adenoviruses. Deletions removing or penetrating from either direction into the conserved sequence inactivated the cloned adenovirus origin. A point mutation within the conserved sequence impaired the adenovirus origin, but point mutations outside the conserved sequence had no effect. These results strongly suggest that the conserved sequence within the first 20 bp of the ITR alone constitutes the adenovirus origin (ori) signal.

The complete sequences of the yeast a mating-type locus, MATa, and of the silent alpha cassette, HML alpha, have been determined. A segment of 642 nucleotides is unique to MATa, and a corresponding segment of 747 nucleotides is unique to MAT alpha. The major mRNAs (a1, a2, alpha 1 and alpha 2) encoded by MATa and MAT alpha have been aligned with the DNA sequence. The a1 mRNA is encoded entirely within the a-specific DNA sequence. The a2 mRNA, which is transcribed divergently from a1 mRNA, is encoded in a region common to both Mata and Mat alpha. The alpha 1 and alpha 2 mRNAs are also transcribed divergently and have their 5' starts about 240 nucleotides apart within the alpha-specific sequence. The amino acid sequences of the MAT proteins have been predicted from the DNA sequences. An unanticipated conclusion is that the a1 protein, containing 148 amino acids, results from readthrough of a UGA at codon 45. Polymorphic forms of the homologous outer segments of the HML alpha, MAT alpha, MATa and HMRa sequences suggest that the boundaries of the segments involved in mating-type switching are immediately adjacent to the a-specific and alpha-specific sequences.

Four virus-specific transcripts have been identified in murine cells infected with Minute-Virus-of-Mice (MVM). These RNAs, 4.8, 3.3, 3.0 and 1.8 kilobases in length, designated R1 to R4 respectively, are all transcribed from the virion (-) strand of DNA and they are all polyadenylated and spliced. The R1 transcript is derived from sequences that reside on the genome between 4.0 and 95 map units (mu). Transcript R2 is composed of exon sequences derived from mu coordinates 4.0-10.0, 40-46 and 48-95. The most abundant RNA, R3, is transcribed from sequences mapping between 40 and 95 mu. All three of these RNAs have a short intron sequence between 46-48 mu removed. The least abundant transcript, R4, has not been mapped precisely, however it hybridizes with all three EcoRI fragments which span the entire 5 kb genome. In vitro transcription of cloned restriction fragments of MVM DNA confirm the existence of functional promoters at map coordinates 4.0 and 39 and sequence analysis of these regions of the viral DNA reveal the characteristic features of RNA polymerase II promoters. These results indicate that MVM DNA encodes two overlapping transcription units with separate promoters near the left end (4.0 mu) and middle (39 mu) of the genome.

The nucleotide sequences of the 3' termini of the DNA from four autonomous rodent parvoviruses have been determined. The terminus of each genome exists as a Y-shaped hairpin structure involving 115 or 116 nucleotides. The sequence of this region of DNA is highly conserved and shows no evidence of internal sequence heterogeneity, a characteristic which is observed in the terminal nucleotide sequence of the helper-dependent, adeno-associated viruses (Berns et al., 1978a). The implications of these results with respect to the models of parvovirus DNA replication are discussed.

DNA fragments isolated from the left end of chromosome III in the yeast Saccharomyces cerevisiae are recognized as telomeres in yeast, since plasmids constructed with such fragments are replicated as linear molecules in yeast. The fragments have a 1.18-kb region homologous with the type X regions at yeast telomeres that contain ARS elements that allow the autonomous replication of plasmids in yeast. The X region contains a functional ARS element including the ARS consensus sequence and a distal 200-bp region that enhances ARS function. Distal to the type X region, the fragments have a region of tandemly repeated DNA sequence defined by the formula 5′-C(1–3)A-3′, and designated as the T region at yeast telomeres. Although the terminus of the chromosome was removed in the procedure used to molecularly isolate the left end of chromosome III, the fragments containing the X and T region are recognized as chromosome ends in yeast. Plasmids with inverted repeats of the chromosome III end fragments are resolved in yeast to produce linear plasmids with telomeres that are similar in length and heterogeneity with the natural left end on chromosome III. Fragments with progressive deletions from the distal end of chromosome III were prepared and used to study the X – T region requirements for distinguishing the fragments as telomeres in yeast.