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Under specified growth conditions, simian virus 40 encapsidated host DNA in a noncircular form free of viral DNA. Two bands of virus particles were present in cesium chloride equilibrium density centrifugation. The host DNA species contained in the upper band was of a lower molecular weight than the DNA present in the mature virus in the lower band.
Simian virus 40 deletion mutants were constructed lacking specifically the intervening sequences for a late viral mRNA. The construction method involved the replacement of portions of the late simian virus 40 genes with the DNA segment from reverse transcription of the viral mRNAs. Restriction endonuclease cleavage and sequence analysis confirmed the precise structure of the mutant DNAs and demonstrated that they contained the genetic information for VP1, including all potential 5' ends for the late viral RNAs. Thus, the primary late transcription product(s) of this mutant should have the structure of functional 16S mRNAs. Complementation analysis as well as immunoprecipitation showed, however, that deletion of the intervening sequences from this mutant prevented the expression of VP1. The nature of this failure appears to be a defect in the posttranscriptional processing of the viral RNA. These results indicate that splicing is an essential function in the biogenesis of certain mRNAs.
Marked differences were found in the susceptibility of human fibroblasts to transformation by simian virus 40 (SV40). Highly susceptible cell strains were derived from patients with diseases associated with chromosomal abnormalities and a high incidence of tumors. In the present study, SV40 transformation-susceptible cell strains were not found to have a generalized increase in viral sensitivity. The differences in transformation frequency among cell strains with whole virus are eliminated by the use of isolated SV40 deoxyribonucleic acid, suggesting that the relative resistance of most cell strains to transformation by whole virus is due to a block at an early step in infection.
Simian virus 40 (SV40), a non-enveloped DNA virus, is transported from the cell surface to the nucleus where virus replication occurs. This pathway of virus uptake involves binding to surface MHC class I molecules, entry via non-coated pits, and subsequent transport to the endoplasmic reticulum (ER). At some stage in this pathway the virus must cross a membrane to reach the cytosol. In the present review, the cellular machinery which the virus has utilized to enter the cell will be examined. In particular, we will consider recent evidence for the involvement of caveolae in the infectious entry step and propose a model involving recruitment of caveolar proteins around the membrane-bound virus. We also speculate that a similar mechanism may have been exploited by bacterial pathogens. The subsequent steps by which SV40 reaches the ER remain unclear but recent evidence suggests that this pathway may be shared with several other proteins that are transported from surface caveolae to the ER.
Simian virus 40 (SV40) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfecta (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (approximately 60%) of the cells were blocked in the G2 + M phase of the cell cycle. At later time intervals, an increase in the G1 population was demonstrated. The two monkey cell lines responded to SV40 virus with an accumulation of cells in the G2 + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV40 virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transforming) cells infected with SV40 virus.
Simian Virus 40 (SV40) induces in "contact-inhibited" tissue culture cells of mouse kidney an abortive infection that leads to the appearance of intra-nuclear SV40-specific tumor (T-) antigen, followed by replication of the mouse-cell chromatin and mitosis, while no viral progeny DNA or capsid protein is produced. Synthesis of "early" SV40-specific RNA ("19S RNA") begins a few hours before the appearance of T-antigen and appears to be switched off after the onset of chromatin replication. As the most simple working hypothesis that can account for the experimental results available, we assume that early SV40 RNA contains information necessary for production of T-antigen and that this antigen (or an unknown early virus-specific function that would simply parallel the appearance of T-antigen) activates or de-inhibits a cellular regulatory element that governs chromosome replication and mitosis. The experimental results agree with the idea that SV40 acts primarily as a mitogen.
Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.
Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.
Simian virus 40 (SV40) binds to MHC class I molecules anywhere on the cell surface and then enters through caveolae. The fate of class I molecules after SV40 binding is not known. Sensitivity of 125I-surface-labelled class I molecules to papain cleavage was used to distinguish internalized class I molecules from class I molecules remaining at the cell surface. Whereas the caveolae-enriched membrane microdomain was found to also be enriched for class I molecules, no internalized papain-resistant 125I-surface-labelled class I molecules could be detected at any time in either control cells or in cells preadsorbed with saturating amounts of SV40. Instead, 125I-surface-labelled class I molecules, as well as preadsorbed 125I-labelled anti-class I antibodies, accumulated in the medium, coincident with the turnover of class I molecules at the cell surface. The class I heavy chains that accumulated in the medium were truncated and their release was specifically prevented by the metalloprotease inhibitor 1,10-phenanthroline. Thus, whereas class I molecules mediate SV40 binding, they do not appear to mediate SV40 entry.
Simian virus 40 early mutants with deletions mapping in the 0.53-0.60 region have been sequenced by the Maxam and Gilbert approach. All these deletions effect the small-t gene. The size of the shortened small-t-related polypeptides produced by several of the mutants has been compared with the molecular weight as deduced from the nucleotide sequence. There was good agreement for the mutants dl890, dl891, and dl2102. For dl2121 and dl2122 the small-t-related protein was considerably larger than expected. It is possible to explain this result on the basis of the nucleotide sequence: the normal splicing event of the small-t mRNA still occurs, but as the deletion shifts the reading frame, translation of the small-t-related polypeptide continues beyond the small-t splice, but in a different reading frame than large-T. Mutants dl883, dl884, and dl2112 have lost one of the small-t splicing boundaries, and no (or minute amonts of) small-t-related protein has been observed in mutant-infected cells. The possible relationship between splicing and transport of polyadenylic acid-containing mRNA from the nucleus to the cytoplasm in vertebrae cells is discussed.
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The simian virus 40 (SV40) mutant dl1055 carries a 55 base pair deletion in the viral early region coding sequences that causes premature termination of large tumour (T) antigen translation resulting in a protein fragment consisting of the amino-terminal 399 residues. The mutation renders the virus defective. We report the characterization of hyb4001, which was isolated as a single plaque following cotransfection of permissive cells with dl1055 DNA and the DNA of a related papovavirus, simian agent 12 (SA12). Hyb4001 arose by two homologous recombination events involving crossovers in regions of 7 and 12 base pairs of perfect homology between the two viruses. Hyb4001 synthesizes a hybrid T antigen with the amino-terminal 382 residues encoded by SV40, residues 383 to 449 encoded by SA12, and the carboxy-terminal 259 residues encoded by SV40.
Simian virus 40 (SV40) was rescued from heterokaryons of transformed mouse and transformed human cells. To determine whether the rescued SV40 was progeny of the SV40 genome resident in the transformed mouse cells, the transformed human cells, or both, rescue experiments were performed with mouse lines transformed by plaque morphology mutants of SV40. The transformed mouse lines that were used yielded fuzzy, small-clear, or large-clear plaques after fusion with CV-1 (African green monkey kidney) cells. The transformed human lines that were used did not release SV40 spontaneously or after fusion with CV-1 cells. From each mouse-human fusion mixture, only the SV40 resident in the transformed mouse cells was recovered. Fusion mixtures of CV-1 and transformed mouse cells yielded much more SV40 than those from transformed human and transformed mouse cells. The rate of SV40 formation was also greater from monkey-mouse than from human-mouse heterokaryons. Deoxyribonucleic acid (DNA) from SV40 strains which form fuzzy, largeclear, or small-clear plaques on CV-1 cells was also used to infect monkey (CV-1 and Vero), normal human, and transformed human cell lines. The rate of virion formation and the final SV40 yields were much higher from monkey than from normal or transformed human cells. Only virus with the plaque type of the infecting DNA was found in extracts from the infected cells. Two uncloned sublines of transformed human cells [W18 Va2(P363) and WI38 Va13A] released SV40 spontaneously. Virus yields were not appreciably enhanced by fusion with CV-1 cells. However, clonal lines of W18 Va2(P363) did not release SV40 spontaneously or after fusion with CV-1 cells. In contrast, several clonal lines of WI38 Va13A cells did continue to shed SV40 spontaneously.
We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.
Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.
The effect of interferon on simian virus 40 (SV40) and adenovirus 2 (Ad2) T antigen synthesis has been examined in cells infected with SV40, with Ad2, and with a nondefective Ad2-SV40 hybrid virus, Ad2(+)ND(4). The induction of SV40 T antigen by SV40 was highly sensitive to interferon, whereas the induction of Ad2 T-antigen by Ad2 was resistant. This difference in interferon sensitivity was also noted in cells simultaneously infected with both viruses. However, the induction of SV40 T antigen by Ad2(+)ND(4), which contains covalently linked SV40 and Ad2 DNAs, was as resistant to interferon as the induction of Ad2 T antigen. This change in the interferon sensitivity of SV40 T antigen synthesis suggests that the expression of at least this portion of the SV40 genetic information in Ad2(+)ND(4) is under Ad2 genetic control. When RNA extracted from Ad2(+)ND(4)-infected cells was examined by means of sequential hybridization with Ad2 DNA, elution, and rehybridization with SV40 DNA, 27% of the SV40-specific RNA was found to be linked to Ad2 RNA. No such linkage was detected in control mixtures of Ad2 and SV40 RNAs. The presence of Ad2 and SV40 nucleotide sequences in the same RNA molecule implies that, in Ad2(+)ND(4) infection, transcription is initiated in the DNA of one virus (Ad2 or SV40) and continues without interruption across the point of junction into the DNA of the other virus. Furthermore, the interferon resistance of Ad2(+)ND(4)-induced SV40 T antigen synthesis suggests that transcription of the genetic information for SV40 T antigen is initiated in a region of Ad2 DNA.
Macrophage migration inhibitory (MIF-like) activity was demonstrated in the supernatant fluids from primary cultures of African green monkey kidney cells infected with simian virus 40 (SV 40) virus. Kidney cell cultures not infected by virus had no MIF activity. Supernatant fluids from continuous cultures of nontransformed and SV 40-transformed human fibroblasts contained MIF-like activity. Productive infection with SV 40 virus results in the production of a lymphokine-like factor, as previously observed in other virus-cell systems, involving mumps virus and Newcast,le disease virus. However, while infection with these paramyxoviruses causes the production of macrophage and neutrophil chemotactic agents as well as an MIF, SV 40 infection does not induce chemotactic factors. The results reported here, taken in conjunction with previous observations by ourselves and others, suggest that the production of lymphokine-like factors (cytokines) may represent a general biologic phenomenon, and that many, if not all, cell types, when appropriately stimulated, may be capable of such activity.
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.