Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope

Nucleus- and mitochondrion-free membranes from hamster lymphocytes transformed by simian virus 40 (SV40), GD248 cells, cause guinea pigs to produce immune sera that reveal the presence in GD248 plasma membranes and mitochondria of two types of glycoprotein that are not detected in membranes of normal lymphocytes [Schmidt-Ullrich, R., Thompson, W. S. & Wallach, D. F. H. (1977) Proc. Natl. Acad. Sci. USA 74, 643-647]. Indirect immune fluorescence of living, SV40-transformed T19 hamster reticulum cells, Balb/c 3T3 mouse fibroblasts, and W18 VA2 human fibroblasts, using the antisera against GD248 membrane, at 4 degrees produced a distinct cell surface fluorescence; however, above 20 degrees , staining at the nuclear perimeter, the SV40 U-antigen reaction, becomes equally prominent. In SV40-transformed cells that had been fixed in cold acetone, as well as in purified GD248 nuclei, thermostable U-antigen staining is dramatic, but there is no reaction for nuclear T-antigen. Rabbit antisera against T19 cells gave immunofluorescence reactions equivalent to those obtained with the antisera against GD248 cells. Normal guinea pig or rabbit sera and cells that had not been transformed by SV40 gave no reaction. Our sera from tumor-bearing hamsters gave only nuclear T-antigen fluorescence. The results indicate the presence of related, SV40-specific antigens in the surface membranes, nuclear envelope, and possibly other intracellular organelles of SV40-transformed cells.     

Monomer molecular weight of T antigen from simian virus 40-infected and transformed cells.

T-antigens from simian virus 40 (SV 40)-transformed and lytically infected cells have been isolated by immunoprecipitation and their molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T-antigen from SV40-transformed mouse and hamster cells has an apparent molecular weight of 94,000 whereas that from several lines of SV40-infected monkey cells is 84,000. In a wheat germ cell-free system, mRNA from either transformed or productively infected cells is translated into a 94,000 species. Experiments with the protease inhibitors L-l-(tosylamide-2-phenyl)ethylchloromethyl ketone HCl and N-alpha-p-tosyl-L-lysylchloromethyl ketone HCl suggest that the 84,000 species of T-antigen found in infected cells is derived from the larger species by proteolytic cleavage. Further, the cleavage pathway probably involves a two-step reaction with an 89,000 intermediate. The biological significance of the two molecular weight forms of T-antigen is unknown, but the possibility that they have different physiological activities is discussed.     

Nature and origin of the RNA associated with simian virus 40 large tumor antigen.

Simian virus 40 (SV40) large tumor (T) antigen isolated from mammalian cells undergoing lytic or transforming infection is associated with small RNA fragments ("T-antigen RNA") that are protected from nuclease digestion. The rather high complexity of the ribonuclease T1 fingerprints of T-antigen RNA suggested that it is mainly derived from cellular heterogeneous nuclear RNAs. In the present study, 5'-32P-labeled T-antigen RNA was hybridized to monkey, mouse, and human Alu and SV40 DNA, and the nucleotide sequence of 37 T1 oligonucleotides was determined. The results suggest that the bulk of T-antigen RNA is derived from noncoding, double-stranded, ordered regions of cellular heterogeneous nuclear RNAs that exhibit sequence homologies with interspersed repetitive elements of the cellular genome. The possible biological implications of these results are discussed.     

Quantitation and characterization of a species-specific and embryo stage-dependent 55-kilodalton phosphoprotein also present in cells transformed by simian virus 40.

A 55-kilodalton (kDal) protein was detected recently in primary cultures of day 12 mouse embryos by immunoprecipitation with serum from simian virus 40 (SV40) tumor-bearing hamsters (T serum), Preliminary evidence suggested that this protein was similar to a cellular 55-kDal protein induced after SV40 transformation of mouse cells. We now show that specific approximately 55-kDal [35S]methionine-labeled proteins precipitate from primary cultures of midgestation mouse, rat, and hamster embryos on addition of T serum or monoclonal antiserum prepared against the SV40-induced mouse 55-kDal proteins. The two-dimensional maps of the [35S]methionine-labeled tryptic peptides of the mouse, hamster, and rat embryo proteins are similar to the maps of the corresponding proteins from SV40-transformed cells. Primary cells from midgestation mouse, hamster, or rat embryos contain one-third to one-half as much 55-kDal protein as a SV40-transformed mouse fibroblast cell and nearly the same amount as F9 mouse embryonal carcinoma cells. The amount of 55-kDal protein is greatly reduced on replating the mouse, rat, or hamster embryo primary cells. The amount of this protein in mouse embryos is dependent on the stage of the embryo. The embryo proteins are phosphoproteins.     

Assignment of the T-antigen gene of simian virus 40 to human chromosome C-7

Hybrid cell clones between mouse cells deficient in thymidine kinase (EC 2.7.1.21) and two different human cell lines transformed by simian virus 40 (SV40) and deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were examined for SV40 tumor (T) antigen(s). Concordant segregation of the gene(s) for SV40 T antigen and human chromosome C-7 was observed in these hybrids. The human chromosome C-7 which contains the gene(s) for SV40 T antigen is preferentially retained by the majority of the hybrid clones tested. When hybrid clones positive and negative for SV40 T antigen, derived from the fusion of SV40-transformed Lesch-Nyhan fibroblasts with mouse cells, were fused with CV-1 permissive cells, SV40-specific V antigen was observed only in the cultures derived from fusion of the hybrid clones positive for T antigen. This result indicates a linkage relationship between human chromosome C-7, SV40 T-antigen gene(s), and SV40 genome(s) integrated in the human transformed cells.     

Resistance of simian virus 40-transformed hamster cells to the cytolytic effect of activated macrophages: a possible factor in species-specific viral oncogenicity.

Simian virus 40 (SV40)-transformed hamster cells were relatively resistant to the lytic effect of activated macrophages from animals with chronic intracellular infections. Conversely, SV40-transformed mouse and rat cells and adenovirus 2-transformed hamster cells were highly susceptible to destruction by tumoricidal activated macrophages. The pattern of resistance or susceptibility of SV40-transformed rodent cells was the same whether activated macrophage effectors were obtained from mice, random-bred hamsters, or the inbred LSH hamsters from which some of the SV40-transformed hamster lines were derived. The results suggest that resistance of transformed cells to macrophage-mediated cytolysis may explain in part the species-specific oncogenicity of this DNA virus.     

Photoreceptor degeneration induced by the expression of simian virus 40 large tumor antigen in the retina of transgenic mice.

Expression of the viral oncogene encoding the simian virus 40 (SV40) large tumor antigen (T antigen) typically promotes tumorigenesis in mammalian cells. To generate transgenic mice that express T antigen in rod photoreceptors, a chimeric construct consisting of a mouse opsin promoter fragment fused to the coding region of SV40 T antigen was generated. Expression of T antigen in the transgenic retina began at early stages of postnatal development concomitant with expression of endogenous opsin. Instead of inducing hyperplasia or tumor formation, T-antigen expression caused a rapidly progressing photoreceptor degeneration. The degeneration was accompanied by sustained DNA synthesis in photoreceptor cells, as evidenced by incorporation of [3H]thymidine and by the appearance of mitotic figures at postnatal day 10, a stage when nontransgenic photoreceptor cells are postmitotic and quiescent. Although transgenic photoreceptor cells undergo S phase and enter mitosis, the consequences of T-antigen expression are not proliferation and tumorigenesis but proliferation and cell death.     

Cytoplasmic transfer of DNA containing simian virus 40 sequences into mouse 3T3 cells.

This paper describes the rare cytoplasmic transmission of defective simian virus 40 (SV40) viral DNA from enucleated cells (i.e., cytoplasts) of the SV40-transformed mouse cell line SVT2 (chloramphenicol-resistant) into cybrid cells formed by fusion of these cytoplasts with BALB/c 3T3 cells (thymidine kinase-deficient). The cybrids were selected in medium containing 1% serum, bromodeoxyuridine, and chloramphenicol. They were identified by their 3T3 chromosome content, by the instability of tumor (T)-antigen expression, by their transformed phenotype, and by their drug resistance. The yield of rare cybrids was about 5 x 10(-7) 0.1% of the yield on medium with 10% serum. The presence of the SV40 genome was detected by the expression of SV40-specific T antigen and confirmed (unpublished data) by hybridization of viral DNA probes with restriction enzyme fragments of nuclear DNAs from cybrid clones. Restriction site mapping (unpublished data) showed that at least 1 kilobase of host flanking DNA on each side of the SV40 DNA was included in the transferred segment. The transforming DNA was not stably integrated initially, as judged by cellular heterogeneity in T-antigen expression. Stable T-antigen-positive and negative subclones were recovered in 10% serum; instability could be retained for at least 30 doublings during growth in 1% serum. The instability is interpreted as evidence of non-integration or unstable integration of the transferred DNA into the host genome. The cytoplasmic transfer is interpreted as evidence that chromosomal fragments or intact chromosomes can be transferred rarely through the cytoplasm in cybrid crosses.     

Electron-Nuclear Double Resonance of a Protein That Contains Copper: Evidence for Nitrogen Coordination to Cu(II) in Stellacyanin

The study of hybrids from three crosses between mouse cells and SV40-transformed human cells have established a positive correlation between the loss of human chromosomes and that of the SV40-induced T-antigen from the hybrid cells. These results, as well as those of other workers, provide strong support for the hypothesis of the integration of the SV40 genome in the chromosomes of transformed cells. Further, it has been shown that hybrid cells which have lost T-antigen are capable of synthesizing this antigen upon infection with SV40, thereby demonstrating that loss of the viral antigen from the hybrid cells is not due to loss of some cellular gene required for the expression of the viral genome. Results of karyological analyses of the hybrid cells argue against the existence of a single specific integration site for the SV40 genome in human cells.     

"Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication.

Simian virus 40 (SV40) induces tumor (T)-antigen formation, chromatin replication, and mitosis in primary mouse kidney cells arrested in G0 phase of the mitotic cycle. The temporal and quantitative relation between these early virus-specific reactions led to the hypothesis that the early SV40 mRNA contains information necessary for T-antigen formation and induction of cellular DNA synthesis. To get direct experimental evidence for this hypothesis, the early strand of SV40 DNA was transcribed in vitro by Escherichia coli DNA-dependent RNA polymerase and the SV40-specific cRNA was transferred by microinjection into epitheloid cells of confluent primary mouse kidney cultures. T-antigen formation and stimulation of DNA synthesis were investigated in the recipient cells. The experimental results obtained agree with the hypothesis that T-antigen is a virus-coded protein and that the early virus-specific mRNA contains information necessary for stimulation of cellular DNA replication in the arrested cells.     

Identification of simian virus 40 tumor and U antigens.

The synthesis and identity of the tumor and U antigens of simian virus 40 (SV 40) have been examined during productive infection in monkey cells, abortive infection in mouse cells, and in SV40-transformed mouse cells by using sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis to analyze [35S]methionine-labeled radioimmune precipitates. The following observations were made: (i) the tumor and U antigenic sites are on the same 94,000, 89,000, and 84,000 molecular weight species detected during productive infection; a 94,000 species made during abortive infection; and a 94,000 species found in transformed cells. (ii) The 94,000 species is relatively unstable compared to the relatively stable 89,000 and 84,000 species produced during productive infection. (iii) The stable 89,000 and 84,000 molecular weight species are differentially extracted from productively infected cells, which suggests an intracellular compartmentation and/or different affinities of these species for cellular substrates. (iv) The 94,000 species synthesized during abortive infection is more stable than the comparable 94,000 species synthesized in transformed cells. (v) Three tsA group mutants overproduce several unstable species of tumor antigen at restrictive temperature.     

Mechanism of interferon action: Simian virus 40-specific early polypeptides synthesized in untreated and interferon-treated monkey kidney cells

The effect of interferon treatment on proteins synthesized in simian virus 40 (SV40)-infected cells in the presence of cytosine arabinoside was investigated. The following results were obtained: (i) In addition to previously described large tumor (T) antigen (94 kilodaltons) and small tumor (t) antigen (19 kilodaltons), a 62-kilodalton polypeptide was immunoprecipitated by SV40 anti-T antiserum from extracts of infected CV-1 and BSC-1 monkey kidney cells and transformed SV3T3 mouse cells. The 94-, 62-, and 19-kilodalton polypeptides were not precipitated with normal serum from extracts of infected cells, and they were not present in extracts of uninfected cells. (ii) The de novo synthesis of the 94-, 62-, and 19-kilodalton tumor antigens was inhibited in CV-1 and BSC-1 cells treated with interferon before infection; total cellular protein synthesis was not significantly affected by interferon treatment. The relative interferon sensitivity of the three polypeptides in lytically infected monkey cells was comparable; by contrast, interferon did not affect their synthesis in transformed mouse cells. (iii) The 62-kilodalton polypeptide was detected in monkey cells infected with the following strains of SV40: tsA30 at both 33 degrees C and 41 degrees C; wt 708, the parent of tsA30; dI 884; and wt 830, the parent of dI 884. The amount of the 62-kilodalton species relative to T antigen was significantly greater in tsA30-infected cells as compared to cells infected with other SV40 strains. (iv) T, t, and 62-kilodalton polypeptides were readily labeled with [(35)S]methionine during a 10-min pulse; in a subsequent chase, the (35)S-labeled 94-kilodalton T antigen was apparently converted to 89- and 84-kilodalton polypeptides but not to either the 62-kilodalton polypeptide species or t antigen. (v) Partial peptide maps suggest that the 62-kilodalton polypeptide and T antigen are closely related. (vi) In addition to the above described 62-kilodalton polypeptide, a 54-kilodalton polypeptide was also detected. However, the 54-kilodalton species appears to be of cellular origin because it was immunoprecipitated with both normal and anti-T antiserum from uninfected and lytically infected cells and from virally transformed cells.     

Antigenic Study of Unfertilized Mouse Eggs: Cross Reactivity with SV40-Induced Antigens

Serum obtained from guinea pigs immunized with unfertilized C57BL/6 mouse eggs was found to be cytotoxic in the presence of complement for eggs obtained from syngeneic and allogeneic mice. The anti-egg serum was not cytotoxic for rat eggs, lymph node cells, methylcholanthrene-induced tumor cells, or 3T3 cells obtained either from syngeneic or allogeneic mice. The anti-egg serum was, however, cytotoxic for SV40-transformed 3T3 cells and C57BL/6 cells. After absorption with SV40-transformed cells, anti-egg serum lost its cytotoxicity for mouse eggs.     

Assignment of the integration site for simian virus 40 to chromosome 17 in GM54VA, a human cell line transformed by simian virus 40.

GM54VA human cells transformed by simian virus 40 (SV40) were fused with peritoneal macrophages obtained from three different mouse strains. All 27 hybrid clones studied were positive for SV40 tumor antigen in 100% of their cells and contained human chromosome 17. Human chromosome 17 was the only human chromosome present in five of the hybrid clones. Fusion of GM54VA cells and either thymidine kinase (EC 2.7.1.75)-deficient mouse or Chinese hamster fibroblasts resulted in the growth in hypoxanthine-aminopterin-thymidine medium of hybrid clones positive and negative for SV40 tumor antigen. Counterselection of the hybrid clones positive for tumor antigen in medium containing 5-bromodeoxyuridine resulted in the growth of hybrid cells that were negative for tumor antigen. These experiments indicate that negative for tumor antigen. These experiments indicate that SV40 is integrated in only one of the two parental human chromosomes 17. Because the genome of SV40 has been assigned to human chromosome 7 in two other SV40-transformed human cell lines, at least two different integration sites for SV40 would seem to be present in human cells: one located in human chromosome 7 and the other located in human chromosome 17.     

Integration and expression of a truncated simian virus 40 early gene fragment in mammalian cells.

The recombinant plasmid p102 based on pBR322 carrying approximately equal to 50% of the replicator proximal early region of simian virus 40 (SV40) DNA, including the viral origin of replication, has been constructed. It lacks a major part of the large tumor (T) antigen 3'-coding region, the T-antigen termination codon, and the polyadenylylation site. The plasmid was transferred together with the herpes simplex virus thymidine kinase (TK) gene as a selectable marker to mouse LTK- cells. TK+ cell clones were isolated and their high molecular weight DNAs were shown by DNA blotting and hybridization experiments to contain the SV40 DNA fragment from the recombinant. In some of these clones, heterogeneous expression of the SV40 DNA fragment could be detected by immunofluorescence while, in control experiments in which a plasmid containing the complete SV40 early DNA region was used, this extensive heterogeneity of T-antigen expression was not observed. RNA . DNA hybridization experiments showed that the SV40-specific RNA of those clones is polyadenylylated. The molecular weight of the T-antigen-related protein coded by p102 corresponded well to the expected coding capacity of the SV40 DNA fragment. Small tumor antigen was not expressed.     

Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.

We describe a rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they are coupled covalently. Specific proteins are detected by autoradiography after sequential incubation with unfractionated, unlabeled specific antiserum and 125I-labeled protein A from Staphylococcus aureus. Antibody and protein A can be removed with urea and 2-mercaptoethanol, and the same paper can be probed again with a different antiserum. An antiserum specific for the simian virus 40 virion proteins VP3 and VP2 has been prepared; it does not crossreact with VP1, as demonstrated by this method. An antiserum raised in rabbits against simian virus 40-transformed rabbit kidney cells is shown to be directed primarily against a periodate-sensitive moiety present in tumor (T) antigen from infected or transformed cells, whereas an antiserum raised in rabbits against large T antigen purified from lytically infected monkey kidney cells by electrophoresis in the presence of sodium dodecyl sulfate [Lane, D.P. & Robbins, A.K. (1978) Virology 87, 182-193] is directed primarily against determinants that are not sensitive to periodate.     

Thermolabile T (tumor) antigen from cells transformed by a temperature-sensitive mutant of simian virus 40.

Partially purified tumor (T) antigen from a strain of Chinese hamster lung cells transformed by wild-type simian virus 40 (SV 40) and either of two temperature-sensitive SV 40 mutants has been studied as a DNA binding protein. The DNA binding activity present in the T-antigen-containing fractions is inhibited by purified hamster anti-T IgG but not by equivalent amounts of nonimmune hamster IgG. T from either wild-type- or tsC219-transformed cells is relatively stable during heating at 44 degrees compared to T prepared from tsA239-transformed cells. These results strongly suggest that T is a product of the SV 40 A gene.