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B Hirt

Publications and source records attributed to B Hirt.

At least 19 recordsLinked to original sources
PubMed1995

The mismatched nucleotides in the 5'-terminal hairpin of minute virus of mice are required for efficient viral DNA replication.

The 5'-terminal sequence in the DNA of the parvovirus minute virus of mice (MVM) is a palindrome. It can form a hairpin, the stem of which is entirely base-paired except for three consecutive unpaired nucleotides which form a bubble. Since this structure is well conserved among different parvoviruses, we examined its importance for viral replication by generating MVM mutants with alterations in this region. A clone of MVMp DNA which contained the entire 3' end and more than half of the 5' palindrome was made. Although it lacked the sequence information to form a wild-type bubble, this DNA was infectious. On transfection into A9 fibroblasts, it gave rise to a virus (MVMs) which had a bubble in its 5' palindrome. The bubble consisted of four mismatched nucleotides in the same location as the unpaired nucleotides of the wild-type palindrome. Apparently, neighboring plasmid sequences were incorporated into the viral DNA, enabling formation of the mismatch. This observation suggested that a bubble is critical for growth of MVM but that its sequence is not. To find out whether MVM lacking a bubble in the 5' palindrome is viable, we made a second clone in which the plasmid sequences incorporated in MVMs were removed. Transfection of this DNA gave rise to a virus (MVMx) in which the nucleotides unpaired in the wild-type hairpin are now fully base-paired. Although MVMx can be propagated, it is defective in comparison with wild-type MVMp; it exhibited about a 50-fold-lower ratio of plaque-forming units to DNA content. In mixed infections, MVMp consistently outgrew the bubbleless MVMx. The rate of accumulation of DNA replication intermediates was lower for MVMx than for the wild-type virus. Quantitative analysis of the 5' termini of replicative form DNA suggested that the ability of MVMx to convert hairpin 5' termini to extended termini is impaired. In contrast, the virus with the altered bubble, MVMs, behaved like the wild-type MVMp in all the assays. We conclude that MVM lacking a bubble in its 5'-terminal DNA hairpin is less infectious than and has a selective disadvantage compared with wild-type MVM. The nucleotide sequence of the bubble is not critical. We provide evidence that the presence of a bubble is necessary for efficient viral DNA replication.

Animals↗
PubMed1991

The minute virus of mice capsid specifically recognizes the 3' hairpin structure of the viral replicative-form DNA: mapping of the binding site by hydroxyl radical footprinting.

The terminal hairpin structures of the DNA of minute virus of mice (MVM) are essential for viral replication. Here we show that the hairpin 3' terminus of MVM replicative-form DNA binds specifically to empty MVM capsids. Binding of the same terminal DNA sequence in its linear double-stranded (extended) conformation was not observed. After heat denaturation and quick cooling of 3'-terminal extended-form fragments, not only the virion strand but also the complementary strand was found to bind to the capsid, presumably because each strand re-formed a similar hairpin structure. No binding affinity for the capsid was found to be associated with hairpin or extended 5' termini or with any other region of the viral DNA. Hydroxyl radical footprinting analyses revealed three protected nucleotide stretches forming a binding site at the branch point of the two 3'-terminal hairpin arms looping out from the DNA stem (T structure). Single base changes within this site did not affect the binding. In band shift experiments, specific binding to the T structure was demonstrated for VPI but not for VP2.

Base Sequence↗
PubMed1990

Nonstructural protein of parvoviruses B19 and minute virus of mice controls transcription.

The genome of the human parvovirus B19 contains a transcriptional promoter (BP06) at map position 6, upstream from the nonstructural protein genes. By cotransfecting HeLa cells with this promoter cloned before the chloramphenicol acetyltransferase (CAT) gene together with a plasmid containing almost the whole B19 genome, we showed that BP06 is transactivated by a B19 gene product. The transactivating viral protein was identified as the nonstructural protein NS-1. NS-1 synthesized in a wheat germ extract specifically stimulates transcription from BP06 in vitro. NS-1 of the minute virus of mice (MVM) activates the analogous MVM promoter, MP04. NS-1, therefore, has a positive feedback effect on the activity of its own promoter. Moreover, NS-1 of MVM activates the human BP06. We have identified, in the genome of B19, a second transcriptional promoter activity at map position 44, before the capsid protein genes. This promoter, BP44, was identified by cloning fragments of B19 DNA upstream of the CAT gene, transfecting the DNA into HeLa cells, and measuring CAT expression. The strength of the BP44 promoter is similar to that of the capsid gene promoter, MP39, of MVM. In (nonpermissive) HeLa cells, the BP44 promoter is not activated by NS-1. Thus, the BP06 promoter apparently does not determine the tissue specificity of B19 virus but BP44 could do so.

Base Sequence↗
PubMed1989

Control region of SV40 minichromosomes is preferentially cleaved by single-strand specific S1 nuclease.

We have analysed S1 sensitivity of SV40 minichromosomes isolated from the nuclei of infected cells at the late stage of infection. We show that a fraction of purified minichromosomes is sensitive towards double-strand cleavage by S1 nuclease. The pattern of specific cleavage reminiscent of that found for subcloned fragment under supercoiling is superimposed upon apparently random double-strand cuts along the entire regulatory region. Therefore, the cleavage sites are not exclusively confined to the regions with the reported alternate DNA conformation.

DNA Restriction Enzymes↗
PubMed1988

Minute virus of mice non-structural protein NS-1 is necessary and sufficient for trans-activation of the viral P39 promoter.

The genome of the autonomous parvovirus minute virus of mice (MVM) is organized in two overlapping transcription units: the genes coding for the two non-structural proteins (NS-1 ad NS-2) are transcribed from a promoter (P04) located at map unit 4, whereas the promoter controlling the capsid protein genes (P39) lies at map unit 39. We studied the effect of viral proteins on the activity of the P39 promoter in vivo. By site-directed mutagenesis we constructed clones encoding only one of the two NS proteins. The activity of the P39 promoter was measured in HeLa or EL-4 cells transfected with these clones, either by an RNase protection assay or by following the expression of a reporter gene, CAT (which codes for chloramphenicol acetyltransferase), placed under the control of this promoter. We found that the P39 promoter of strain MVMi is activated in trans by a viral gene product, and evidence to suggest that NS-1 is the only viral gene product responsible for this trans-activation. We also determined that the mechanism of trans-activation is very rapid, since all species of viral mRNAs appear together in non-synchronized infected EL-4 cells within a 2 h interval.

Amino Acid Sequence↗
PubMed1988

Characterization of the cell type-specific determinant in the genome of minute virus of mice.

Two strains of minute virus of mice (MVM) show different host cell specificities. The prototype strain MVM(p) grows in fibroblasts, whereas the immunosuppressive variant MVM(i) grows in T lymphocytes. In this study, we have mapped on the viral genome a cell type-specific determinant: it is located between 69 and 85 map units in a region coding for the viral capsid proteins. The DNA of MVM(p) does not replicate in lymphocytes. MVM(i) cannot help MVM(p) grow in lymphocytes; thus the determinant acts in a cis fashion. We did not detect viral mRNA during a restrictive infection of lymphocytes with MVM(p). However, when the same cells were transfected with cloned DNA, both MVM(p) and MVM(i) DNAs were transcribed with the same efficiency from both promoters and the RNA was processed normally. Therefore, the specificity determinant is not a cell type-specific enhancer.

Cell Line↗
PubMed1987

A transcriptional promoter of the human parvovirus B19 active in vitro and in vivo.

The human parvovirus B19 causes aplastic crises in sickle cell anemia patients and the disease erythema infectiosum. So far, it has not been possible to grow B19 virus in cultured cells. Here we report the use of in vitro transcription in HeLa cell extracts and transient expression of cloned DNA transfected into HeLa cells to detect and map a strong transcriptional promoter on the B19 genome. The promoter is located near the left end of the B19 genome, at position 6 map units in the clone pYT103 (approximately 280 bp upstream of the first HindIII site), and directs transcription to the right. These results suggest that the strictly limited host range of B19 does not operate at the level of transcription from the promoter at the left end of the genome.

DNA, Viral↗
PubMed1986

The genes for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) are tandemly arranged on chromosome 17 of the mouse.

We have isolated clones containing the gene for tumor necrosis factor (TNF-alpha) from a mouse genomic library. Four out of five clones containing the TNF-alpha gene also hybridized to a human lymphotoxin (TNF-beta) probe. We constructed a restriction enzyme cleavage map of a 6.4 kb region from one of the genomic clones. From partial sequencing data and hybridizations with exon-specific oligonucleotide probes, we conclude that this region contains the mouse TNF-alpha and TNF-beta genes in a tandem arrangement, that they are separated by only about 1100 bases, and that their intron-exon structure is very similar to that seen in man. We probed genomic blots of DNA from human/mouse hybrids containing single mouse chromosomes for the presence of the mouse TNF genes. The results show that the genes are located on mouse chromosome 17, which also contains the major histocompatibility complex. Therefore, both the mouse and the human TNF genes are tandemly arranged and located on the same chromosome as the MHC.

Animals↗
PubMed1986

Pathogenicity of fibroblast- and lymphocyte-specific variants of minute virus of mice.

We tested two strains of the minute virus of mice (MVM) for pathogenic effects and patterns of infection in laboratory mice. The two strains differ in their ability to infect differentiated cultured cells: the prototype virus, MVMp, infects only fibroblasts, while its variant, MVMi, is restricted to lymphocytes. We find that neither strain has any demonstrable effects on the T-cell function of mice infected as adults. In contrast, MVMi, but not MVMp, is able to induce a runting syndrome accompanied by mild immune deficiencies upon the infection of newborn mice. After neonatal infection, MVMi spreads to many organs, and the presence of viral replicative form DNA is evident in nucleic acid hybridization experiments. In contrast, replication of MVMp can be detected only by the seroconversion of infected animals. Newborn mice that grow abnormally as a result of MVMi infection also have low circulating antibody titers to the virus. This phenomenon may be a consequence of the lymphotropism of MVMi.

Animals↗
PubMed1986

A precise map of splice junctions in the mRNAs of minute virus of mice, an autonomous parvovirus.

We have determined the exact splicing patterns of the mRNAs of the minute virus of mice by a combination of cDNA sequencing and S1 nuclease protection analysis. There are four virus-specific mRNA species, each coding for one of the four polypeptides identified by in vitro translation. The R1 mRNA comprises sequences from nucleotide approximately 200 to 2281 and from 2378 to approximately 4800 and codes for the NS1 protein. The R2 mRNA is derived from nucleotides approximately 200 to 515, 1991 to 2281, and 2378 to approximately 4800 and codes for the NS2 protein. Between nucleotides 1991 and 2281, the coding sequence for NS2 overlaps that of NS1, but in a different reading frame. R3 covers nucleotides approximately 2007 to 2281 and 2378 to approximately 4800 and codes for VP2. The fourth species, R3', differs from R3 by using an alternative splice donor and acceptor in the region around 47 map units (nucleotide 2400); it extends from nucleotide approximately 2007 to 2317 and from 2400 to approximately 4800 and almost certainly codes for VP1. The R2 transcript is unusual in that the intron that was removed from it (nucleotides 516 to 1990) starts with GC rather than the canonical GU. With the exception of the splice acceptor at position 2378, which is found only in rodent parvoviruses, the splice junctions are highly conserved among autonomous parvoviruses. These results show that minute virus of mice, like other small DNA viruses, uses multiple strategies to compress the coding information for several viral proteins into a short (5,104 nucleotide) genome.

Base Sequence↗
PubMed1985

DNA sequence comparison between two tissue-specific variants of the autonomous parvovirus, minute virus of mice.

We have determined the complete nucleotide sequence of the DNA of the immunosuppressive variant of the parvovirus minute virus of mice (MVMi) and compared it to the published sequence (12) of the fibroblast-specific strain (MVMp). We have found 175 differences between the two viruses, most of which affect single nucleotides. Despite these differences, the genomic organization of MVMp and MVMi is identical. There are 29 amino-acid changes between the putative viral gene products of MVMi and MVMp, 16 of which are conservative. We discuss the possibility that the differential tissue-specificity of the two variants is linked to differences within the non-transcribed region near the 5' end of the viral genomes.

Animals↗
PubMed1981

Characterization of an immunosuppressive parvovirus related to the minute virus of mice.

We have characterized an immunosuppressive parvovirus related to the minute virus of mice (MVM). The parvovirus, MVM(i), grew efficiently on the murine lymphoma cell line EL-4 and not on the A-9 strain of L-cells which is a host for the prototype MVM. MVM(i) was immunosuppressive for allogeneic mixed leukocyte cultures, inhibiting the generation of cytolytic T lymphocytes. MVM had no effect on mixed leukocyte cultures. MVM and MVM(i) particles were similar in buoyant density, sedimentation rate, appearance in the electron microscope, and polypeptide composition. We present restriction enzyme maps of the DNAs of MVM and MVM(i) which show that they are closely related. Out of 109 restriction endonuclease cleavage sites (representing together about 10% of the nucleotide sequence), 86 sites were shared by MVM and MVM(i), whereas 22 sites were absent from one of the two viruses. MVM(i) DNA had an apparent deletion of about 60 nucleotides relative to MVM, located near the 5' terminus of viral DNA.

Animals↗
PubMed1981

Inhibition of T cell-mediated functions by MVM(i), a parvovirus closely related to minute virus of mice.

A purified preparation of MVM(i), a murine parvovirus closely related to minute virus of mice (MVM), was found to inhibit various functions mediated by murine T cells in vitro. Addition of MVM(i) virus to secondary allogeneic mixed leukocyte cultures resulted in the inhibition of both lymphocyte proliferation (3H-thymidine incorporation) and the generation of cytolytic T lymphocyte activity but not interferon production. MVM(i) virus also inhibited the growth and cytolytic activity of several cloned, long-term Lyt-2+ cytolytic T cell lines. Furthermore, the antigen-induced proliferative responses of parasite- (Leishmania) specific Lyt-1+ T cells in vitro was abrogated by the addition of MVM(i) virus to the culture. Finally, the suppression of an in vitro antibody response to SRBC by MVM(i) virus was the result of the inhibition of T helper cells required for the B cell response. These suppressive effects were specific for MVM(i); parallel studies in which the prototype MVM parvovirus was used showed no significant inhibition in the various systems tested.

Animals↗
PubMed1975

Folding of the DNA double helix in chromatin-like structures from simian virus 40.

Relaxed circular, covalently closed simian virus 40 DNA molecules were associated with the four histones that are present in virions. In electron micrographs the resulting complexes appear twisted, with globular structures (nucleosomes) along the DNA. Incubation with an untwisting extract converts the twisted complexes to relaxed structures. Extraction of the DNA from the relaxed complexes yields supercoiled molecules. The number of superhelical turns in these molecules corresponds to the number of nucleosomes per DNA molecule in the complexes.

Animals↗