Rescue of infectious virus from permissive monkey cells containing simian virus 40 DNA fragments.

Permissive TC7 cells were separately transfected with simian virus 40 (SV40) EcoRI/Hap II A (74% genome) DNA fragments and EcoRI/Hap II B (26% genome) DNA fragments in the presence of DEAE-dextran. Fusion of the progeny of recipient cells receiving the A fragment, TC7 (SV40/74) cells, with TC7 (SV40/26) cells, which had received the B fragment, resulted in SV40 rescue. TC7 (SV40/74 + 26) cells, which had simultaneously received both complementary subgenomes, either spontaneously produced SV40 upon subculture or yielded virus upon treatment with iododeoxyuridine. In addition, fusion of rat cells containing the EcoRI/Hap II A fragment with TC7 (SV40/26) cells resulted in SV40 rescue. Cytopathology, V-antigen production, neutralization, and electron microscopy were parameters used to verify that the rescued virus was SV40. No infectious virus was produced when the combinations of cells fused did not total a complete SV40 genome equivalent.     

Virion-mediated transfer of SV40 epigenetic information

In eukaryotes, epigenetic information can be encoded in parental cells through modification of histones and subsequently passed on to daughter cells in a process known as transgenerational epigenetic regulation. Simian Virus 40 (SV40) is a well-characterized virus whose small circular DNA genome is organized into chromatin and, as a consequence, undergoes many of the same biological processes observed in cellular chromatin. In order to determine whether SV40 is capable of transgenerational epigenetic regulation, we have analyzed SV40 chromatin from minichromosomes and virions for the presence of modified histones using various ChIP techniques and correlated these modifications with specific biological effects on the SV40 life cycle. Our results demonstrate that, like its cellular counterpart, SV40 chromatin is capable of passing biologically relevant transgenerational epigenetic information between infections.     

Virion-mediated transfer of SV40 epigenetic information

In eukaryotes, epigenetic information can be encoded in parental cells through modification of histones and subsequently passed on to daughter cells in a process known as transgenerational epigenetic regulation. Simian Virus 40 (SV40) is a well-characterized virus whose small circular DNA genome is organized into chromatin and, as a consequence, undergoes many of the same biological processes observed in cellular chromatin. In order to determine whether SV40 is capable of transgenerational epigenetic regulation, we have analyzed SV40 chromatin from minichromosomes and virions for the presence of modified histones using various ChIP techniques and correlated these modifications with specific biological effects on the SV40 life cycle. Our results demonstrate that, like its cellular counterpart, SV40 chromatin is capable of passing biologically relevant transgenerational epigenetic information between infections.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Random integration of SV40 in SV40-transformed, immortalized human fibroblasts

We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.     

Influence of SV40 Genome on the Replication of an Adenovirus-SV40 "Hybrid" Population.

Feldman, L. A. (Baylor University College of Medicine, Houston, Tex.), J. L. Melnick, and F. Rapp. Influence of SV40 genome on the replication of an adenovirus-SV40 "hybrid" population. J. Bacteriol. 90:778-782. 1965.-Replication of a type 7 adenovirus-SV40 hybrid population in primary African green monkey kidney cells was accompanied by the formation of SV40 tumor antigen, adenovirus antigens, and cytopathic changes characteristic of adenovirus infection. Prior infection of the cultures with SV40 stimulated replication of nonintegrated adenovirus 7 but did not enhance the replication of the hybrid virus. These results suggest that the population of the adenovirus-SV40 hybrid studied contains many particles carrying SV40 information. Replication of SV40 virus was not enhanced by co-infection with nonintegrated adenovirus 7 or with the adenovirus-SV40 hybrid. Cytosine arabinoside strongly inhibited replication of the adenovirus-SV40 hybrid population in African green monkey kidney cells. Enhanced replication of nonintegrated adenovirus 7 by SV40 was blocked by cytosine arabinoside; this block could be reversed by 2-deoxycytidine or deoxycytidine triphosphate.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. II. Biochemical Evidence for Genetic Exchange

The genome of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 rescued by passage on transformed permissive monkey lines (see accompanying paper [Y. Gluzman et al., J. Virol. 24:534-540, 1977]) was analyzed by restriction endonuclease cleavage mapping to obtain biochemical evidence that the rescue of the ts phenotype results from recombination with the resident SV40 genome of the transformed cell. It was demonstrated that the endonuclease R. HaeIII cleavage site, which is located at 0.9 map unit in the standard viral genome (and which is in the proximity of the known map position of the tsD lesion), is missing in the DNAs of the parental tsD202 virus and of three independent revertants of tsD202. In contrast, this cleavage site was shown to be present in the DNAs of four out of five independently derived rescued D202 populations and in the DNA of the SV40 strain, 777, used to transform the monkey cells. Comparison of the endonuclease R. Hin(II + III) cleavage patterns of SV40 strain 777 DNA and tsD202 DNA revealed differences in the electrophoretic mobilities of Hin fragments A, B, and F. However, the corresponding Hin fragments from all four rescued D202 genomes were identical in their mobilities to those of tsD202 DNA, indicating that these regions of the rescued D202 genome are characteristic of the tsD202 parent. We conclude, therefore, that the genome of the rescued D202 virus is a true recombinant, since it contains restriction endonuclease cleavage sites characteristic of both parents, the endogenous resident SV40 genome of the transformed monkey cells and the exogenous tsD202 mutant.     

Fusion-mediated injection of SV40-DNA : Introduction of SV40-DNA into tissue culture cells by the use of DNA-loaded reconstituted Sendai virus envelopes

Sendai virus envelopes can be solubilized by non-ionic detergents such as Triton X-100. Removal of the detergent from a supernatant containing the solubilized viral envelope glycoproteins results in the formation of reconstituted fusogenic viral envelopes. When SV40-DNA is added to the reconstitution system, it is trapped within the viral envelope. Incubation of SV40-DNA-loaded Sendai virus envelopes with permissive cells (CV1 and TC7 cells) resulted in fusion-mediated injection of the trapped DNA, as was demonstrated by the ability of the injected cells to synthesize SV40-T-antigen. Quantitative estimation revealed that up to 20% of the injected cells were able to synthesize T-antigen. Loaded viral envelopes were able to inject SV40-DNA and to promote synthesis of T-antigen also in cells which are resistant to infection by intact SV40 viruses, such as F1' 1-4 cells. In addition, it is shown that reconstituted envelopes of Sendai virus are able to transfer membrane fragments from SV40 receptor-positive into SV40 receptor-negative cells, such as F1' 1-4 cells. After implantation of SV40 receptors, the F1' 1-4 cells became susceptible to infection by intact SV40 viruses.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: II. Detection and Characterization of Simian Virus 40 Pseudovirions

Purified simian virus 40 (SV40) virions, grown in primary African green monkey kidney cells labeled prior to infection with (3)H-thymidine, contain a variable quantity of (3)H-labeled deoxyribonucleic acid (DNA). This DNA is resistant to deoxyribonuclease, sediments at 250S, and is enclosed in a particle that can be precipitated with SV40-specific antiserum. DNA-DNA hybridization experiments demonstrate that this (3)H-labeled component in purified SV40 virions is cellular DNA. When this (3)H-labeled DNA is released from purified virus with sodium dodecyl sulfate, it has an average sedimentation constant of 14S. Sedimentation through neutral and alkaline sucrose gradients shows that this 14S DNA is composed of a collection of different sizes of DNA molecules that sediment between 11 and 15S. As a result of this size heterogeneity, SV40 virions containing cellular DNA (pseudovirions) have a variable DNA to capsid protein ratio and exhibit a spectrum of buoyant densities in a CsCl equilibrium gradient. Pseudovirions are enriched, relative to true virions, on the lighter density side of infectious SV40 virus banded to equilibrium in a CsCl gradient. Little or no cellular DNA was found in purified SV40 virus preparations grown in BSC-1 or CV-1 cells.     

Analysis of sera from SV40-immunized and tumor-bearing hosts for blocking activity and antibody-dependent cellular cytotoxicity.

The role of serum factors in tumor immunity to cells transformed by PARA-(defective SV40)-adenovirus 7 was investigated. It was found that sera from SV40-sensitized hosts did not block the specific cytotoxicity of SV40-sensitized spleen cells for PARA-7 cells. However, such sera could collaborate with nonsensitized spleen cells to produce specific killing. This activity could be absorbed out by PARA-7 cells but not by cells transformed by cytomegalovirus. The activity of sera from hamsters bearing tumor isografts depended upon when, after transplantation, the specimens were obtained. Sera collected greater than or equal to 10 days after grafting completely blocked immune spleen cell cytotoxicity and did not mediate target cell killing in the presence of normal spleen cells. Sera obtained at an earlier time, i.e., 3 to 6 days after transplantation, consistently were active in the antibody-dependent cellular cytotoxicity test and exhibited reduced or no blocking of antibody-independent cellular cytotoxicity. Thus, there appears to be an inverse correlation in the capacity of serum from tumor bearing hosts to block effector cell cytotoxicity and mediate antibody-dependent cellular cytotoxicity.     

Integration and excision of SV40 DNA from the chromosome of a transformed cell

The single insertion of SV40 DNA present in the genome of the 14B line of transformed rat cells has been cloned in procaryotic vectors. Analysis of the clones reveals a complex arrangement of viral sequences in which a small tract of DNA is inverted with respect to the major insertion. The nucleotide sequences at the two junctions show sharp transitions between cellular and viral sequences. The sequences which flank the viral insertion have been used as probes to clone the corresponding genomic sequences from the DNA of untransformed rat cells. Analysis of the structure of these clones shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration. When 14B cells are fused with uninfected simian cells a heterogeneous set of low molecular weight superhelical DNAs containing viral sequences is generated. These have been cloned in procaryotic vectors and their structures have been analyzed. All of them span the origin of SV40 DNA replication and are colinear with various segments of the integrated viral DNA and its flanking sequences. The shorter molecules contain part of the integrated viral genome and cellular sequences from one side of the insertion. They were therefore generated by recombination between the viral DNA and its flanking cellular sequences. The longer molecules contain cellular sequences from both sides of the insertion as well as an entire copy of the integrated viral DNA. They were therefore generated by recombination between the flanking cellular sequences. These results argue strongly against the involvement of specific excision enzymes, and rather are discussed in terms of a model involving replication of the integrated viral DNA followed by recombination for release of integrated viral sequences.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.     

Simian Virus 40-Host Cell Interactions. I. Temperature-Sensitive Regulation of SV40 T Antigen in 3T3 Mouse Cells Transformed by the ts*101 Temperature-Sensitive Early Mutant of SV40

BALB/3T3 and Swiss/3T3 mouse cells transformed at permissive temperature (33 C) by the early temperature-sensitive mutant of simian virus 40 (SV40), ts(*)101, exhibited a temperature-dependent modulation of SV40 tumor (T) antigen as assayed by immunofluorescence. The percentage of T antigen-positive nuclei in ts(*)101 transformed cells was reduced at restrictive temperature (39 C) when compared to 33 C and to wild-type SV40 transformed cells at either 33 C or 39 C. The percentage of T antigen-positive nuclei in ts(*)101 transformed cells returned to the 33 C control level when the cells were shifted from 39 to 33 C. The ts(*)101 transformed cells could be superinfected with wild-type, but not ts(*)101, virions at 39 C as assayed by an increase in T antigen-positive nuclei.     

Interaction of SV40 T-antigen with homologous and heterologous DNA

Using the DNA filter binding assay, the effects of ionic strength and pH on SV40 T-antigen interaction with viral DNA were studied. The apparent association constants for T-antigen binding to SV40 DNA in Scatchard coordinates in the presence of 40 mM NaCl are equal to 0.67 . 10(6) M-1 (pH 6.0) and 0.86 x 10(7) M-1 (pH 7.4). These data indicate that the interaction between T-antigen and SV40 DNA is more specific at pH 7.4. The coincident values of association constants for T-antigen binding to viral and cellular DNAs (Ka = 0.9 x 10(7) M-1 for cellular DNA) at pH 7.4 and the absence of competition between the two DNA species upon binding with T-antigen suggest that viral and cellular DNAs possess similar sites for T-antigen binding. Denatured DNA competes with viral DNA only at pH 6.0, when the T-antigen--SV40 DNA interaction is less specific.     

Reversibility of malignant transformation as affected by the oncogenic virus SV40. II. The characteristics of virus-induced revertant clones

Fifteen revertant clones exhibiting contact inhibition, one of the typical characteristics of normal cells, were studied after treatment of spontaneously transformed Chinese hamster fibroblasts with SV40. The clones proved to be partial revertants, as regards to other properties of the normal phenotype--loss of the ability to grow in a medium with a low serum content and anchorage-dependence. Viral DNA was detected in all revertant clones. The expression of T-antigen--the product of viral oncogene, was observed in 13 of 15 revertants analyzed. The study of SV40 "rescued" from several revertants in permissive monkey cells has shown that the virus is non-defective. In 7 clones, reversion was accompanied with polyploidization. In the cases, reversion could be due to changes in the balance between oncogenes and suppressor genes (anti-oncogenes). The possibility of induction by SV40 of mutations in anti-oncogenes suppressing the expression of both cellular and viral oncogenes is discussed. It is suggested that reversion to the normal phenotype in clones with a near-diploid karyotype could result from such virus-induced suppressor mutations.     

Structure of simian virus 40-adeno-associated virus recombinant genomes.

The structures of recombinant genomes formed by recombination between simian virus 40 (SV40) and adeno-associated virus 2 (AAV) DNAs after either DNA cotransfection or coinfection by virions were characterized. Two types of structures were found. Group A structures, found after cotransfection and in one of seven recombinants arising from coinfection, represented a simple deletion of SV40 sequences replaced by a slightly shorter AAV sequence. Group B structures were found in six of seven recombinants arising after virion coinfection. All contained either the left or right terminal sequences (approximately 250 to 450 bases) of the AAV genome adjacent to the SV40 origin of DNA replication. Only 350 to 650 bases (including the origin) remained of the SV40 sequence. The joined SV40-AAV sequences were present in the recombinant genome as a tandem repeat of a size that can be packaged into SV40 capsids.