Viral Minichromosomes Research Articles

Host range transforming gene of polyoma virus plays a role in virus assembly.

Polyoma virus host range transforming (hr-t) mutants are blocked in virion assembly. In normal 3T3 cells, a nonpermissive host, these mutants synthesize 30-40% as much viral DNA and 80-100% as much capsid proteins as does wild-type virus and yet produce only 1-2% as much infectious virus. Intermediates in virion assembly have been followed by [3H]thymidine incorporation. hr-t mutants synthesize 95S replicating minichromosomes, which accumulate as 75S forms. However, the latter fail to undergo efficient transition to 240S virion structures. This block in encapsidation is overcome in permissive hosts such as primary baby mouse kidney (BMK) epithelial cells. The block in assembly of 240S particles is accompanied by a failure to induce a series of acidic isoelectric forms of the major capsid protein, VP1. Multiple species of post-translationally modified VP1 are seen by two-dimensional gel electrophoresis in wild-type virus-infected cells. These acidic VP1 subspecies are decreased 6- to 10-fold in hr-t mutant-infected 3T3 cells but are produced in normal amounts when the same mutants infect BMK cells. When 3T3 cells are coinfected with hr-t mutant and wild-type viruses, normal amounts of the VP1 subspecies are present, and hr-t mutant viral DNA is efficiently packaged into virions. These studies demonstrate an important role of the hr-t gene of polyoma virus in virus assembly. Specifically, we propose that VP1 is a target for hr-t gene-controlled modification and that modified forms of VP1 are essential for encapsidation of viral minichromosomes.

In vitro premature termination in SV40 late transcription.

Nuclear extracts and viral transcribing minichromosomes were prepared from SV40-infected cells and incubated in vitro with [alpha-32P]UTP under conditions which allow the elongation of preinitiated RNA chains. Sucrose gradient lysis of the transcription mixtures revealed two populations of SV40-specific RNA: elongating chains that remain associated with the viral minichromosomes, and, at the top of the gradient, small free RNA detached from the template and hybridizing exclusively to the promoter-proximal region of SV40 DNA. This free RNA was shown by polyacrylamide gel electrophoresis to comprise essentially a 94 nucleotide species, which could, however, at high UTP concentration, be elongated a further few nucleotides before terminating. These results thus show that the actively transcribing minichromosomes provide a sytem in which the attenuated RNA can be released from the template. Moreover, this is the first demonstration of specific in vitro termination of polymerase B transcription. The conditions which lead to transcription termination are discussed.

The DNase I sensitivity of Xenopus laevis genes transcribed by RNA polymerase III.

Since the initial discovery that the DNase I sensitivity of the globin genes in different cell types correlates with globin gene expression, this relationship has been shown to hold true for a variety of genes, including the genes for ovalbumin, conalbumun, alpha- and beta-globin in chicken, several heat-shock proteins in Drosophila, the r-chromatin of Tetrahymena and the viral polyoma minichromosome. Although genes transcribed by RNA polymerases I and II have been studied extensively, the genes transcribed by RNA polymerase III have not. We have therefore investigated the DNase I sensitivity of transfer RNA (tRNA) and oogenetic 5S RNA genes in the liver and erythocyte nuclei of Xenopus laevis. The oogenetic 5S genes are not transcribed in any known somatic cell, and tRNA genes are transcribed in the hepatocyte but are inactive in the erythrocyte. We show here that, although in these two cell types the correspondence between DNase I sensitivity and gene transcription holds good for globin and the ribosomal genes, the tRNA and oogenetic 5S genes are DNase I sensitive in both liver and erythrocyte nuclei. Thus for the genes transcribed by polymerase III the correspondence of sensitivity and expression breaks down.

Absence of nucleosomes in a histone-containing nucleoprotein complex obtained by dissociation of purified SV40 virions

Reduction of disulfide bonds involving the major capsid protein with dithiothreitol and removal of the calcium ions by EGTA disrupts the simian virus 40 virions. This process yields normal circular viral minichromosomes containing the four core histones and traces of the capsid proteins at pH values higher than 8.5. However, when carried out at pH 7.5, this procedure yields nucleoprotein complexes that contain both histones and the viral structural proteins. These pH 7.5 complexes appear as circular structures with a mean of 93 ± 17 beads with a diameter of 7 nm and no visible nucleosomes when observed by electron microscopy. In contrast to the compaction of the viral DNA in minichromosomes, the length of these beaded structures is roughly the same as free DNA. We suggest that VP1, the major capsid protein, can act as a nucleosome unfolding agent in neutral pH and low ionic strength.

Quantitation of transcribing native simian virus 40 minichromosomes extracted from CV1 cells late in infection.

Simian virus 40 transcriptional complexes could be extracted from CV1 cells late in infection and separated from the bulk of inactive viral chromatin. Sucrose gradient sedimentation, cesium sulfate equilibrium density gradient centrifugation, and electron microscopy indicated that the viral transcriptional complexes corresponded to at least 0.5 to 1% of the viral minichromosomes extracted, about 5,000 copies per cell.

Role of Chromatin Structure, Histone Acetylation, and the Primary Sequence of DNA in the Expression of SV40 and Polyoma in Normal or Teratocarcinoma Cells

Publisher Summary The extensive knowledge about the viral genome and its minichromosomes permits a more detailed analysis of the nature of the regulating elements governing the expression of the viral genome at the level of the DNA primary sequence and at the level of the chromatin. The region containing the initiation sites for late, and probably for early, transcription has a unique conformation in the viral minichromosomes. This region is devoid of nucleosomes in 5% to 20% of the viral chromosomes, as detected by the electron microscopy and by DNase I digestion of nuclei isolated from infected cells. The chapter discusses on analyzing the SV40-transformed rat cells in an attempt to probe the structure of the integrated viral genome and its regulatory regions in these cells. It is clear, from both sequence comparison and transcription studies in vitro, that specific sequences are responsible for “promoter” recognition by eukaryotic RNA polymerases and their cofactors. The sequence studies of polyoma mutants that grow on embryonal carcinoma cells suggest that the promoters recognized in a differentiated cell may differ from those recognized by an embryonic cell. This may be analogous to the modifications of the transcription machinery at both the enzyme and DNA levels after phage SPOl infection of Bacillus subtilis. Embryonic development and differentiation may thus be accompanied by the changes in RNA polymerase II or its cofactors and by the changes in the sequence of the initiation sites recognized on the cellular genome. The control of gene expression by both the primary DNA sequence and the structure of chromatin may permit more refined and tighter control of gene expression in eukaryotic cells.

In situ study of SV40 virus DNA in lytic infection by mild loosening of nucleoproteins.

We have studied SV40 (simian virus40) nucleoprotein in permissively infected monkey kidney cell cultures (CV1) by a procedure which does not require the isolation of the SV40 chromosomes. Treatment of the cells by a low ionic strenght medium containing Photo flo produces a mild loosening of nucleoproteins, and permits the in situ study in ultrathin sections of virus components and their relationships with host cell chromatin. RNP and DNP could be distinguished by uranyl-EDTA-lead staining (for RNP) and by DNase digestion. SV40 DNA was observed as circular molecules, either free or connected with either RNP fibrils or virus capsids. These three aspects were interpreted, respectively, as viral minichromosomes, transcription of virus genome and partially encapsidated virus DNA. During encapsidation a few virus particles appear to be bound to host chromatin. Many, if not all, seemingly mature viruses, singly or in small linear clusters, are also aligned on host chromatin. Some of these observations were corroborated by the Miller spreading technique. They are consistent with a role for the host cell chromatin in the production of nuclear viruses.

Escherichia coli RNA polymerase in vitro mimics simian virus 40 in vivo transcription when the template is viral nucleoprotein.

We have used a low-salt detergent-free extraction procedure on cells infected with simian virus 40 to obtain viral nucleoprotein late after infection. Addition of EScherichia coli RNA polymerase and ribonucleotide triphosphates to the viral minichromosomes permitted transcription of RNA from viral templates. This synthesis was initiated predominantly within a fragment of DNA spanning 0.67 to 0.76 map unit on the genome. The synthesis from this region proceeded primarily along the "late" strand in a clockwise direction. These results were in contrast to the synthesis obtained with naked viral DNA in which initiation occurred on other regions of the genome and from which transcription proceeded counterclockwise along the early strand. These findings indicate that the nucleoprotein template or factors tightly associated with it may be responsible for site(s) and strand selection in transcription of simian virus 40.

Benzo[ a]pyrene metabolism in cells productively infected with Simian virus 40

The metabolism of benzo[ a]pyrene (BP) by cell cultures and cell-free extracts of the monkey kidney cell line CV-1 was studied in uninfected and Simian virus 40 (SV40) infected cells. Metabolites formed were separated by high-pressure liquid chromatography (HPLC) and quantified by liquid scintillation techniques. The profiles of metabolites formed by uninfected and SV40 infected cells were similar except that SV40 infected cell cultures metabolized BP at an increased rate relative to uninfected cells. In addition, SV40 infected cell cultures and cell-free extracts produced an unknown compound which eluted between the 3-hydroxybenzo[ a]pyrene and BP fractions. This material does not have a retention time characteristic of any of the known metabolites of BP. Labelled BP and/or its metabolites were bound to the viral DNA and histone components of intracellular viral minichromosomes as well as the viral DNA and proteins of mature virions.

Efficient transcription of a compact nucleoprotein complex isolated from purified simian virus 40 virions.

Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis-N-N'-tetraacetic acid and dithiothreitol. The compact nucleo-protein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP(1) and VP(2) in addition to the proteins found associated with the viral minichromosome, i.e., VP(3) and histones H(2)A, H(2)B, H(3), and H(4). Our results suggest that the association of VP(1), VP(2), or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of Escherichia coli RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the "early" SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP(1) or VP(2) or both enhances the transcriptional activity and suggest that these "late" viral proteins may play a role in the regulation of expression of the SV40 genome.

Staphylococcal nuclease makes a single non-random cut in the simian virus 40 viral minichromosome

When compact simian virus 40 (SV40) minichromosomes are treated with staphylococcal nuclease at 0 °C under limit-digest conditions, about one-third of the minichromosomes remain resistant to nuclease, a third of them are nicked, while the remaining third suffer one and only one double-stranded cut. Results show that each cleaved minichromosome is cut only once and afterwards becomes resistant to further fragmentation. This is in marked contrast to the action of staphylococcal nuclease at 37 °C, which leads to a rapid fragmentation of all minichromosomes to oligo- and mononucleosomes. The SV40 linear DNA III produced by low-temperature nuclease digestion of minichromosomes was redigested with single-cut restriction endonucleases. By this mapping procedure it was determined that the location of the staphylococcal nuclease cut is neither unique nor random; it occurs at a number of discrete sites on the DNA, half of all cuts being concentrated at the origin of replication and nearby in the “late” portion of the SV40 genome. Control experiments have shown that when staphylococcal nuclease digests naked SV40 DNA at 0 °C it does not “hesitate” after the first cut. Although initial cuts in the purified DNA are non-random in location, their distribution is quite different from that generated by a low-temperature nuclease digestion of compact SV40 minichromosomes. Possible interpretations of these results are discussed in view of the recent finding that a specific region of the SV40 genome is uniquely exposed in the minichromosome (Varshavsky et al., 1978, 1979; Scott & Wigmore, 1978).

The template of the isolated native simian virus 40 transcriptional complexes is a minichromosome

Native simian virus 40 (SV40) transcriptional complexes containing active DNA-dependent RNA polymerase B enzymes that have initiated synthesis in vivo were extracted by salt from nuclei of infected cells late in infection. These complexes can synthesize RNA chains longer than the viral genome under conditions that do not irreversibly alter the structure of the viral chromatin. The RNA synthesized in vitro allowed the separation of most of the viral transcriptional complexes from transcriptionally inactive SV40 minichromosomes. Density studies indicate that about the same amount of protein is associated with both the bulk of the viral minichromosomes and the transcriptional complexes. Parallel shifts in the sedimentation of bulk minichromosomes and transcribing complexes at increasing ionic strength and after nucleosome “exchange” strongly suggest that the actively transcribed viral genome has a structure similar to that of the bulk of viral chromatin. In agreement with the biochemical results, electron microscopy studies strongly suggest that the template for late viral transcription has a minichromosome-like structure.

SV40 viral minichromosome: preferential exposure of the origin of replication as probed by restriction endonucleases

Isolated SV40 minichromosomes [1-3] were treated with different single-cut restriction endonucleases to probe the arrangement of nucleosomes in relation to the SV70 DNA sequence. While Eco RI and Bam HI each cut 22-27% of the SV40 minichromosomes under limit-digest conditions, Bgl I, which cuts SV40 DNA at or very near the origin of replication [4,5], cleaves 90-95% of the minichromosomes in a preparation. Similar results were obtained with minichromosomes which had been fixed with formaldehyde before endonuclease treatment. One possible interpretation of these findings is that the arrangement of nucleosomes in the compact SV40 minichromosomes is nonrandom at least with regard to sequences near the origin of DNA replication.

Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure

We report two new findings bearing on the "supranucleo-somal" level of the structure of the Simian Virus 40 minichromosome. I) Isolated SV40 minichromosome which contains all five histones including HI/I/ exists in solution under approximately physiological ionic conditions as a compact roughly spherical particle approximately 300 A in diameter which is capable of fitting within the virus capsid. In spite of such a compact conformation of the minichromosome individual nucleosomes can be readily visualized within the particle. Compact state of SV40 minichromosome depends on both the presence of histone HI and maintenance of approximately physiological ionic strength of solution (micron approximately 0.15). Removal of HI results in a conversion of the compact minichromosomes into an extended (circular beaded) structure. 2) The compact form of the SV40 minichromosome in contract to its circular beaded form is virtually completely resistant to staphylococcal nuclease, strongly suggesting that in particular nuclease-sensitive parts of the internucleosomal DNA regions are not exposed on the outside of the compact SV40 minichromosome. On the other hand, DNase I which is known to attack both inter-and intranucleosomal DNA in the chronatin /2,3/ readily digests the compact form of the SV40 minichromosome. Possible models of the compact minichromosome and implications for higher order structures of the cellular chromatin are discussed.

Minichromosome of simian virus 40: presence of histone HI

In contrast to conclusions of previous studies /I-3/ claiming the absence of histone HI from the SV40 and polyoma viral minichromosomes we have found that a preparation of purified SV40 minichromosomes does contain histone HI. The content of HI in relation to other four histones in the SV40 minichromosomes is close to that in the cellular chromatin. Histone HI in the isolated SV40 minichromosomes is bound apparently to internucleosomal DNA stretches as was shown already for HI in the cellular chromatin /4/. In addition it was found that more than 90% of the purified SV40 minichromosomes migrated as a single discrete deoxyribonucleoprotein band upon agarose gel electrophoresis.