Deletion mutants which affect the nuclease-sensitive site in simian virus 40 chromatin.

A short segment of simian virus 40 (SV40) chromatin on the late side of the origin of replication is hypersensitive to nuclease cleavage. The role of DNA sequence information in this nuclease-sensitive feature was examined by constructing deletion mutations in this region. Deletions were introduced into the inserted segment of in(Or)-1411 (a viable, partially duplicated variant of SV40), and nuclease sensitivity of the inserted segment was compared with that of the unaltered sequences in their normal location in the viral genome. Extended deletions (118 to 161 base pairs) essentially abolished nuclease sensitivity within the inserted segment. Shorter deletions (21 to 52 base pairs) at separate locations retained the nuclease-sensitive feature. In some short-deletion mutants nuclease susceptibility was substantially reduced. We conclude that more than one genetic element in this region contributes to the organization of the nuclease-sensitive feature and that the SV40 72-base repeat is not, in itself, sufficient signal for this feature.     

DNA replication and chromatin structure of simian virus 40 insertion mutants.

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.     

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.     

Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.

A portion of the nucleoprotein containing viral DNA extracted from cells infected by simian virus (SV40) is preferentially cleaved by endonucleases in a region of the genome encompassing the origin of replication and early and late promoters. To explore this nuclease-sensitive structure, we cleaved SV40 chromatin molecules with restriction enzymes and digested the exposed termini with nuclease Bal31. Digestion proceeded only a short distance in the late direction from the MspI site, but some molecules were degraded 400 to 500 base pairs in the early direction. By comparison, BglI-cleaved chromatin was digested for only a short distance in the early direction, but some molecules were degraded 400 to 450 base pairs in the late direction. These barriers to Bal31 digestion (bracketing the BglI and the MspI sites) define the borders of the same open region in SV40 chromatin that is preferentially digested by DNase I and other endonucleases. In a portion of the SV40 chromatin, Bal31 could not digest through the nuclease-sensitive region and reached barriers after digesting only 50 to 100 base pairs from one end or the other. Chromatin molecules that contain barriers in the BglI to MspI region are physically distinct from molecules that are open in this region as evidenced by partial separation of the two populations on sucrose density gradients.     

Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40.

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.     

Ultraviolet irradiation inhibits encapsidation of simian virus 40 chromatin.

During normal maturation and majority of pulse-labeled simian virus 40 DNA progresses from chromatin to previrions and virions within 5 h. UV light inhibits this progression. In heavily irradiated cultures (108 J m-2) most of the simian virus 40 DNA synthesized immediately before irradiation remains as chromatin for at least 5 h. This inhibition of maturation seems to be a result of the inhibition of protein synthesis. The data suggest that the pool of proteins required for maturation is sufficient to convert one-third of the simian virus 40 DNA molecules labeled in a 10-min pulse (at 33 h postinfection) from chromatin to previrions and virions and is exhausted within 1 h.     

Chromatin assembly. Relationship of chromatin structure to DNA sequence during simian virus 40 replication

The accessibility of five specific DNA sequences to six different single site restriction endonucleases was evaluated in replicating and mature simian virus 40 chromosomes isolated by three different methods. Electron microscopic and gel electrophoretic analysis of the DNA digestion products demonstrated that DNA accessibility in chromatin was established within 400 base pairs of replication forks and remained essentially unchanged during production of mature chromosomes and their subsequent re-entry into the replication pool. Saturating amounts of each enzyme reproducibly cut a fraction of the chromosomes, ranging from 13 to 49%. This is consistent with a nearly random phasing of chromatin structure. Examples in which all chromosomes were either cleaved or intact were never observed. Although variation in the accessibility of DNA sites near the origin of replication could be interpreted as preferred phasing in about 25% of the chromosomes, the finding that two isoschizomers, Hpa II and Msp I, did not cut chromosomes to the same extent precludes an unambiguous interpretation of the extents of cleavage of individual restriction enzymes. Since the extent of DNA cleavage observed at each restriction site was essentially indistinguishable in replicating as compared to mature chromosomes, the accessibility of DNA sequences near the origin is not obviously related to replication. Furthermore, the accessibility of DNA sites on one arm of a single replication fork was the same as the homologous sites on the other arm, consistent with a nearly random phasing of chromatin structure on both arms. This suggests that chromatin assembly occurs independently on the 2 sibling molecules of a single replicating chromosome.     

Excision of integrated simian virus 40 DNA involving homologous recombination between viral DNA sequences

We have investigated the structure of simian virus 40 (SV40) DNA integrated into the genome of transformed mouse mKS-A cells. We have identified at least six independent integration units containing intact or truncated SV40 DNA sequences. One integration unit was isolated from a genomic mKS-A cell library and investigated by restriction enzyme analysis and partial nucleotide sequencing. This integration unit contains one apparently intact SV40 genome flanked on both sides by truncated versions of the SV40 genome. One of the flanking elements contains a large deletion in the SV40 "late" region and an abbreviated SV40 "early" region. This element was efficiently excised and mobilized after fusion of mKS-A to COS cells. The excision products invariably included the entire SV40 early region even though they were derived from an integrated element lacking this part of the SV40 genome. An analysis of this discrepancy led to the conclusion that the early region sequences were acquired by homologous recombination and, furthermore, that homologous excisional recombination was clearly preferred over non-homologous recombination.     

Intracellular localization of viral polypeptides during simian virus 40 infection.

African green monkey kidney cells infected by simian virus 40 were analyzed by immunofluorescence techniques for the nature and the time course of the appearance of viral polypeptides during infection. Reagents used in the study were anti-Vpl sera and affinity-purified anti-Vpl immunoglobulin G, anti-Vp3 sera, antivirus (anti-V) sera, and anti-tumor antigen sera. The results are summarized as follows. (i) Three types of staining, nuclear, perinuclear, and perinuclear accompanied by cytoplasmic staining, were observed in infected cells in reaction with anti-vpl antibody. In addition, a highly structured staining was observed at the periphery of nuclei of infected cells late in infection. (ii) In reaction with anti-Vp3 serum, the staining was confined within nuclei of cells throughout infection. (iii) Vp1 and Vp3 antigens seem to occupy different spacial regions of the nuclear area in cells. (iv) Vp1 and Vp3 antigens were expressed simultaneously during infection. (v) Centriolar staining observed early in infection paralleled the appearance of tumor (T-) antigen until 24 h after infection, after which time the frequency of positive centriolar staining decreased as infection progressed. (vi) T-antigen was first expressed at about 8 h after infection, and Vp1 and Vp3 antigens were first expressed at about 20 h after infection.     

Two types of deletion within integrated viral sequences mediate reversion of simian virus 40-transformed mouse cells.

Simian virus 40 (SV40) DNA insertions from SV40-transformed mouse cell line W-2K-11 and its revertants M18, M31, and M42 were cloned. W-2K-11 cells contain 1.5 copies of the SV40 sequences in a partially tandem duplicated form. The endpoints of the viral sequences at the virus-host junctions are located very close to those reported by others, indicating that there are some preferred sites for integration and rearrangement in SV40 sequences. One flanking cellular sequence is a long stretch of adenine and thymine with repeated AAAT, and the other is a stretch of guanine and cytosine with repeated CCG. There are patchy homologies between the flanking cellular sequences and the corresponding parental SV40 sequences. The sequences around both junctions were retained in all the revertants, whereas most of the internal SV40 sequences coding for large T antigen were deleted. The coding sequences for small T antigen are intact, and small T antigen was expressed in all the revertants. The fragments cloned from M18 and M42 were identical and 3.9 kilobases of SV40 sequences were deleted. The parental SV40 sequences around the deletion site have sequences capable of forming a secondary structure which might reduce the effective distance between the two regions. The SV40 DNA retained in M31 is colinear with SV40 virion DNA, and a unit length of SV40 DNA was deleted within the SV40 sequences present in W-2K-11 cells. These results indicated that two types of deletion occurred during the reversion, one between homologous sequences and the other between nonhomologous sequences.     

Structural changes in simian virus 40 chromatin as probed by restriction endonucleases.

The structure of simian virus 40 (SV40) chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of three different single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard to specific nucleotide sequences within the population of chromatin molecules. However, the extent of digestion was found to be strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the simian virus 40 (SV40) DNA I in chromatin was converted to linear SV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01 M), an additional 20 to 30% of the DNA was cleaved. These results suggest that at 100 mM NaCl only the DNA between nucleosomes was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions became available for cleavage. Surprisingly, when SV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragment, whereas only a small fraction (9 to 15%) received two or more cuts. Instead, the principal digest fragment was full-length linear SV40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after HpaI digestion, it was found that this site was not unique. Nevertheless, all sites wree not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest that the initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We propose that this may be accomplished by either "winding" of the internucleosomal DNA into the body of the nucleosome, or as suggested by others, by successive right-hand rotation of nucleosomes.     

Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases.

Simian virus 40 (SV40) chromatin isolated from infected BSC-1 cell nuclei was incubated with deoxyribonuclease I, staphylococcal nuclease or an endonuclease endogenous to BSC-1 cells under conditions selected to introduce one doublestrand break into the viral DNA. Full-length linear DNA was isolated, and the distribution of sites of initial cleavage by each endonuclease was determined by restriction enzyme mapping. Initial cleavage of SV40 chromatin by deoxyribonuclease I or by endogenous nuclease reduced the recovery of Hind III fragment C by comparison with the other Hind III fragments. Similarly, Hpa I fragment B recovery was reduced by comparison with the other Hpa I fragments. When isolated SV40 DNA rather than SV40 chromatin was the substrate for an initial cut by deoxyribonuclease I or endogenous nuclease, the recovery of all Hind III or Hpa I fragments was approximately that expected for random cleavage. Initial cleavage by staphylococcal nuclease of either SV40 DNA or SV40 chromatin occurred randomly as judged by recovery of Hind III or Hpa I fragments. These results suggest that, in at least a portion of the SV40 chromatin population, a region located in Hind III fragment C and Hpa I fragment B is preferentially cleaved by deoxyribonuclease I or by endogenous nuclease but not by staphylococcal nuclease.Complementary information about this nuclease-sensitive region was provided by the appearance of clusters of new DNA fragments after restriction enzyme digestion of DNA from viral chromatin initially cleaved by endogenous nuclease. From the sizes of new fragments produced by different restriction enzymes, preferential endonucleolytic cleavage of SV40 chromatin has been located between map positions 0.67 and 0.73 on the viral genome.     

Infectious linear DNA sequences replicating in simian virus 40-infected cells.

A new class of linear duplex DNA structures that contain simian virus 40 (SV40) DNA sequences and that are replicated during productive infection of cells with SV40 is described. These structures comprise up to 35% of the radioactively labeled DNA molecules that can be isolated by selective extraction. These molecules represent a unique size class corresponding to the length of an open SV40 DNA molecule (FO III), and they contain a heterogeneous population of DNA sequences either of host or of viral origin, as shown by restriction endonuclease analysis and nucleic acid hybridization. Part of the FO III DNA molecules contain viral-host DNA sequences covalently linked with each other. They start to replicate with the onset of SV40 superhelix replication 1 day after infection. Their rate of synthesis is most pronounced 3 days after infection when superhelix replication is already declining. Furthermore, they cannot be chased into other structures. At least a fraction of these molecules is infectious when administered together with DEAE-dextran to permissive cells. After intracellular circularization, superhelical DNA FO I with an aberrant cleavage pattern accumulates. In addition, tumor and viral capsid antigen are induced, and infectious viral progeny is obtained. Infection of cells with purified SV40 FO I DNA does not result in FO III DNA molecules in the infected cells or in the viral progeny. It is suggested, therefore, that these FO III DNA molecules are perpetuated within SV40 virus pools by encapsidation into pseudovirions.     

The arrangement of simian virus 40 sequences in the DNA of transformed cells.

High molecular weight DNA, isolated from eleven cloned lines of rat cells independently transformed by SV40, was cleaved with various restriction endonucleases. The DNA was fractionated by electrophoresis through agarose gels, denatured in situ, transferred directly to sheets of nitrocellulose as described by Southern (1975), and hybridized to SV40 DNA labeled in vitro to high specific activity. The location of viral sequences among the fragments of transformed cell DNA was determined by autoradiography. The DNAs of seven of the cell lines contained viral sequences in fragments of many different sizes. The remaining four cell lines each contain a single insertion of viral DNA at a different chromosomal location. The junctions between viral and cellular sequences map at different places on the viral genome.     

Subcellular distribution of viral structural proteins during simian virus 40 infection.

The amounts of simian virus 40 structural polypeptides Vp1, Vp2, and Vp3 in different subcellular fractions at various times after lytic infection were determined by a quantitative immunoblotting procedure. Simian virus 40-infected cells were lysed with a buffer containing Nonidet P-40 to yield a soluble fraction. The Nonidet P-40-insoluble fraction was further fractionated in the presence of deoxycholate and Tween 40 to yield a soluble fraction (cytoskeletal) and an insoluble fraction (Nuc), which is primarily cell nuclei. At 33 h postinfection, the majority of viral structural proteins was found in the cell nucleus, whereas, at 48 to 65 h postinfection, Vp1 was distributed evenly among all cell fractions and Vp2 and Vp3 were found predominantly in the cytoskeletal and Nuc fractions. Thus, not all of the viral polypeptides synthesized in the cytoplasm migrated into the cell nucleus. Throughout infection, the molar ratio (Vp3/Vp2) was rather constant in all subcellular fractions, indicating that the synthesis or processing or both of Vp2 and Vp3 are coordinately regulated. The molar ratio of Vp1/(Vp2 + Vp3) varied among the fractions. The Vp1/(Vp2 + Vp3) molar ratio in the soluble fraction varied during the course of infection; however, constant ratios were maintained in the cytoskeletal and Nuc fractions. Thus, the mechanism which controls the movement of Vp1 to different compartments of the cell appears to be different from that of Vp2 and Vp3. The Vp1/(Vp2 + Vp3) value in the Nuc fraction was similar to the ratio found in virus particles. The constant molar distribution of Vp1, Vp2, and Vp3 in the Nuc fraction throughout infection suggests that there is a specific mechanism which regulates the transport of viral structural proteins. These results support the hypothesis that the structural proteins of simian virus 40 are transported into the cell nucleus in precise proportions.     

Presence of free viral DNA in simian virus 40-transformed nonproducer cells.

Extracts from several simian virus 40 (SV40)-transformed nonproducer cells were prepared by the hot-phenol procedure normally used to extract cellular RNA. These extracts contained SV40 infectious units. Part of the infectious units were identified as SV40 form I DNA molecules. The results of reconstruction experiments suggest that SV40 form I DNA is extractable by the hot-phenol procedure because of its fast renaturation rate. The significance of the presence of free viral DNA in nonproducer transformed cells is discussed.