Erratum

Simian virus 40 (SV40) large tumor (T) antigen was isolated from SV40-infected permissive and -transformed cells by immunoprecipitation with anti-T sera and purified by electrophoresis on solubilizable SDS-polyacrylamide gels. This method proved to be a rapid and efficient procedure for purifying T antigen as 74% of the immunoprecipitated T antigen was subsequently recovered. The purified, SDS-denatured large T antigen retained its ability to react with anti-T antibody and to immunize mice against a challenge of syngeneic SV40 tumor cells, indicating that the antigenic sites present on the large T antigen molecule that induce a tumor rejection response in vivo are resistant to denaturation by SDS.     

SV40-transformed cells express SV40 T antigen-related antigens on the cell surface

SV40 tumor antigen (T-Ag)-related antigens were detected serologically on the surface (surface T) of living SV40-transformed human and mouse monolayer cells by an ¹²⁵I-protein A binding assay. In immunofluorescence analysis, these cells were negative for surface T. However, on mKSA, a SV40-transformed mouse cell line grown in suspension or on SV40-transformed human and mouse monolayer cells put into suspension, surface T could be visualized by immunofluorescence microscopy. The antisera used in these experiments were raised in rabbits with purified, SDS-denatured SV40 T-Ag or came from hamsters bearing SV40 tumors. Both types of antisera had in common high titers against SV40 T-Ag (?1:1000). All these antisera were negative on normal cells or on polyoma virus-transformed cells. The specificity of both antisera for SV40 T-Ag-related binding sites on the surface of SV40-transformed cells were demonstrated by an ¹²⁵I-IgG blocking assay in which preincubation of the cells with rabbit anti-T-Ag serum inhibited the binding of hamster SV40 tumor serum to the cell surface by about 85%. These results demonstrate the expression of T-Ag-related antigens on the surface of living cells and, therefore, support the hypothesis that SV40 T-Ag-related antigens participate in the formation of the SV40-specific tumor transplantation antigen (TSTA).     

Identification of new polypeptide species (48-55K) immunoprecipitable by antiserum to purified large T antigen and present in SV40-infected and -transformed cells.

New polypeptide species with molecular weights between 48K and 55K can be immunoprecipitated with serum against purified 94K T antigen (T Ag) of SV40 as well as with antitumor serum. These related species have been separated by SDS-polyacrylamide gel electrophoresis into a spectrum with predominating bands characteristic of each cell line examined. The SV40-infected cell lines examined are the following: SV40-infected TC-7 and Vero cells, and the SV40-transformed lines SV3T3, VLM, SV28, and SV80. The 48K–55K species have not been observed in the immunoprecipitates of uninfected cell extracts. The purified 48K species, excised from gels, can be specifically reimmunoprecipitated with the anti-94K T Ag serum. The possibility that the 48K–55K species may be in vitro proteolytic products of the 94K T Ag has been excluded by a variety of experiments involving the use of mixed extracts, the use of inhibitors of proteolysis, and comparison of their methionine-containing tryptic peptides. Deletion mutants of SV40 mapping between 0.54 and 0.59 map units do not affect the appearance or size of these new species. The SV40 mutant tsA58 produces a 55K species which is very stable at high and low temperatures, suggesting that the mutation does not affect the new antigen. The results suggest that the 48K–55K species may originate either as host-coded species (perhaps induced by the virus) that share determinants with T Ag or perhaps as SV40-encoded species sharing only very little of the amino acid sequence of 94K T Ag.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IV. Inhibition by human interferon of expression of SV40 TrAg in SV40-infected monkey cells.

Human interferon inhibits the synthesis of SV40 transplantation rejection antigen (TrAg) in SV40-infected but not in SV40-transformed monkey cells. The synthesis of SV40 tumor antigen as detected by the indirect immunofluorescence test and the large and small T antigens as detected by the immunoprecipitation with sera from tumor-bearing hamsters and electrophoresis in SDS-gels was similarly affected in SV40-infected monkey cells. These results suggest that the induction of SV40-specific TrAg in the cytolytic cycle depends upon a viral, rather than a host, message.     

A subclass of simian virus 40 T antigen with a high cell surface binding affinity

SV40 virus-infected and -transformed cells express large T antigen on the cell surface (surface T). In the present study the cell surface binding properties of T antigen extracted from SV40-transformed cells were investigated. Only small amounts of T antigen with tight cell surface binding properties were efficiently removable by absorption on living cells from the majority of T antigen detectable in cell extracts. As shown in immunofluorescence microscopy both native surface T and experimentally in vitro cell surface bound T antigen were stained in similar microcluster patterns. Comparative SDS-polyacrylamide gel electrophoretic analysis indicated that T antigen extracted from SV40-transformed cells and in vitro cell surface bound T antigen had the same apparent molecular weight of approximately 90,000 da. A quantitative 125I-protein A binding assay using antisera directed against purified T antigen demonstrated that a metal-ion chelating agent (EDTA) or hypertonic salt solutions were unable to remove surface T or in vitro cell surface bound T antigen from living cells. In contrast, both antigens could be solubilized by detergents. Moreover, both types of cell surface associated T antigens seemed to be metabolically stable. Altogether, one can postulate a minor subclass of T antigen with a tight binding affinity to the cell surface of living cells. According to these properties this experimentally membrane bound subclass, as well as native surface T, seem to belong to the class of integral rather than peripheral membrane proteins.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.     

Domains of simian virus 40 large T-antigen exposed on the cell surface

The orientation of SV40 large T on the surface of SV40-transformed mouse cells and of human cells infected with nondefective adenovirus 2 SV40 hybrid viruses has been studied. Using antibodies against a synthetic peptide corresponding to a region of 11 amino acids at the carboxyterminus of large T, the surface of formaldehyde-fixed SV40-transformed cells could be specifically stained by indirect immune fluorescence. Staining was inhibited by an excess of the peptide. These data suggest that the carboxyterminus of large T is exposed on the surface of formaldehyde-fixed cells. Antibodies against the carboxyterminus of large T also stained the surface of cells infected with the hybrid viruses Ad2⁺ND1, Ad2⁺ND2, and Ad2⁺ND4. Thus, the carboxytermini of the SV40-specific proteins synthesized in hybrid virus-infected cells are also exposed on the cell surface. When analyzed with an antiserum against purified denatured large T, which among many other determinants also recognizes the large T carboxyterminus, surface fluorescence was observed in cells infected by all three hybridviruses. The surface fluorescence of Ad2⁺ND1-infected cells, expressing an SV40-specific protein of 28 K, and Ad2⁺ND2-infected cells expressing SV40-specific proteins of 42 K and 56 K molecular weight, was completely inhibited by carboxyterminal peptide. However, the surface fluorescence of Ad2⁺ND4-infected cells, expressing SV40-specific proteins up to nearly full size large T, was unaffected by carboxyterminal peptide. Our data suggest that a major portion of large T, located between a region near the carboxyterminus and a region corresponding to the aminoterminus of the 56 K protein, is not exposed on the surface of hybridvirus-infected cells. However, some parts of the aminoterminal one-third of large T appear to be exposed again. We conclude that SV40 large T on the surface of SV40-transformed cells is oriented in a specific manner, suggesting that it is specifically associated with the plasma membrane.     

Tumor-specific transplantation antigen is expressed during SV40 lytic infection with wild-type and tsA mutant viruses.

The expression of SV40 tumor-specific transplantation antigen (TSTA) in SV40-infected monkey cells has been assayed using detergent extracts of cell membranes and cytosol to immunize mice prior to an SV40 ascites-tumor challenge. TSTA is expressed during infection of CV-1 cells by wild-type SV40 and by a temperature-sensitive A mutant. Following infection by the tsA mutant, TSTA [like the tumor antigen (TA) but unlike viral DNA, capsid proteins, and mature virus] appears both at restrictive (40.5°) as well as at permissive (33°) temperatures. Kinetic studies show that the initial appearance of TSTA and TA is concurrent.     

The differential effect of interferon on T antigen production in simian virus 40-infected or transformed cells.

The ability of interferon (IF) to inhibit T antigen (Ag) production in simian virus 40 (SV40)-infected or transformed cells was studied primarily through the use of immunoprecipitation followed by gel electrophoresis and autoradiography. Addition of IF to monkey cells prior to or subsequent to inoculation with SV40 resulted in an inhibition in the amount of T Ag that was synthesized late in infection. In contrast when a similar experiment was performed with a is mutant of SV40, tsA58, which does not replicate at the nonpermissive temperature, there was no inhibition in the amount of immunoprecipitable T Ag when IF was added at 30 hr postinfection at 40.5°. The effect of IF on an integrated versus nonintegrated genome within the same cell population was studied in an SV40-transformed mouse cell, H6-15, which is temperature sensitive for the transformed phenotype and for expression of T antigen. In shift-down experiments it was shown that the reappearance of SV40 T Ag was insensitive to the addition of IF whereas superinfection of these same cells with polyoma virus resulted in a dose-dependent inhibition of polyoma T Ag infection. An SV40-transformed mouse cell line (nonpermissive) and two SV40-transformed human cell lines (semipermissive) were passaged in the presence of IF for four generations. Approximately the same amount of labeled T Ag could be immunoprecipitated from IF-treated compared to control mouse cultures whereas, there was a marked decrease in the amount of newly synthesized T Ag in IF-treated human cultures. All these results are compatible with the hypothesis that IF affects differentially the expression of early viral genes whether the viral DNA is integrated or not integrated.     

Immunoselection of simian virus 40 large T antigen messenger RNAs from transformed cells

The messenger RNAs (m-RNAs) coding for the simian virus 40 (SV40) large tumor antigen were substantially purified by immunoprecipitation of polysome fractions from SV40-transformed cells. The level of SV40 T antigen m-RNA in the transformed cell used in this study was estimated at 0.05% of the total population of polyadenylated m-RNAs and a several-hundred-fold purification of this m-RNA was achieved by employing antibodies to SV40 large T antigen purified by either of two methods. The procedures described in this communication should be particularly useful for the immunoselection and purification of low-abundance m-RNAs or when the antigen is not available in large quantities.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). V In vitro demonstration of SV40 TrAg in SV40 infected nonpermissive mouse cells by the lymphocyte mediated cytotoxicity assay.

The development of SV40-specific transplantation antigen (TrAg) on the surface of nonpermissive mouse cells infected with SV40 was demonstrated using a sensitive in vitro lymphocyte mediated cytotoxicity assay. The in vitro lymphocyte mediated ⁵¹Cr release assay was shown to be specific for the detection of SV40 TrAg. SV40 TrAg was detected 24 hr after infection of mouse cells with SV40 and high levels of TrAg expression persisted for as long as 96 to 120 hr after virus infection. The development of TrAg on the surface of SV40-infected mouse cells correlated with the synthesis of tumor or T antigen in the nucleus of infected cells. The synthesis and the expression of TrAg at the surface of SV40-infected mouse cells may be an important step in the development of immunological resistance of the cell mediated type in an SV40-inoculated host leading to the elimination of stably transformed cells.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) III. Involvement of SV40 gene A in the expression of TrAg in permissive cells.

Temperature-sensitive (ts) mutants of simian virus (SV40) which map in the early region of the SV40 genome were used to determine the role of the viral genome in the expression of SV40-specific transplantation rejection antigen (TrAg). The results indicated that tsA mutants (1612, 1637, 7, and 28) did not induce the expression of SV40-TrAg at the surface of infected permissive African green monkey kidney cells (TC-7) at 41° but did induce the expression of TrAg at the permissive temperature (33°) in TC-7 cells. Wild-type SV40 and late SV40 temperature-sensitive mutants (tsBC1602, tsBC1606, tsB8, and tsC219) induced SV40-TrAg in TC-7 cells at nonpermissive and permissive temperatures with equal efficiency. One of the mutants belonging to complementation group D (tsD1601) was defective in inducing SV40-TrAg at 41°. Kinetic studies indicated that SV40-TrAg appears by 18 hr after infection at 41° and 38 hr post-infection at 33°, paralleling closely the synthesis of T antigen. The synthesis of immunoreactive T antigen in TC-7 cells infected with tsA mutants at nonpermissive temperature did not correlate with the inability of tsA mutants to express TrAg at nonpermissive temperature. We conclude that the expression of TrAg in SV40-infected cells depends upon normal functioning of the A gene.     

Cell surface binding simian virus 40 large T antigen becomes anchored and stably linked to lipid of the target cells

Previously a new small subclass of SV40 large T antigen with a high-binding affinity to living target cells was characterized (J. Lange-Mutschler and R. Henning, 1983, Virology 127, 333-344.) In the present study the external binding process, particularly the tight linkage of T antigen to lipid of the target cells, was analyzed. Extraction of SV40-transformed target cells (SV80) first by sonification yielded approx 80% of [35S]methionine-labeled T antigen (mechanical extract). A further 20% was obtained by treatment of cellular debris with hydroxylamine (hydroxylamine extract). As shown by an 125I-protein A radioimmunoassay, hydroxylamine extracts contained significantly higher amounts of cell surface binding T antigen. Correspondingly, after incubating [3H]palmitic acid-prelabeled target cells (HeLa) with unlabeled extracts, predominantly T antigen from hydroxylamine extracts became 3H labeled by the target cells, dependent on metabolic or enzymatic conditions. 3H-labeled T antigen became unlabeled after treatment with hydroxylamine indicating a covalent ester linkage between cell surface-bound T antigen and lipid of the target cells. The cell surface localization of in vitro acylated T antigen was demonstrated by mild trypsin digestion of living target cells. These results strongly support the idea about a novel mechanism by which a minor subclass of T antigen after being bound to the cell surface becomes covalently linked to lipid of the living cell.     

An immunochemical investigation of SV40 T antigens. 1. Production properties and specificity of rabbit antibody to purified simian virus 40 large-T antigen.

Large quantities of a species of T antigen with an apparent molecular weight of 84,000 have been isolated from monkey kidney cells infected with SV40 by using the protein A Antibody Adsorbent (P.A.A.) technique and preparative SDS-polyacrylamide gel electrophoresis. The purified polypeptide was found to be immunogenic, inducing a specific antibody response in a rabbit. The resulting antiserum was 10 times as potent as a hamster anti-tumor serum and reacted with native as well as SDS- and DTT-treated T antigens from SV40-transformed or lytically infected cells. It failed to show any reaction with T antigen from polyoma-infected cells and showed similar specificity to antitumor serum obtained from hamsters which had been inoculated with cells of an SV40-transformed, virus-free cell line. In both cases two distinct polypeptides, large T (84,000 and 94,000) and small t (19,000) were precipitated from extracts of SV40-transformed or lytically infected cells. The rabbit antiserum was shown to be capable of specifically precipitating small-t antigen in the absence of large-T antigen and therefore these two polypeptides must share common antigenic determinants. A radioimmunoassay showed large-T antigen to be very heat stable in direct contrast to earlier results obtained using the complement fixation test. The reasons for this discrepancy and its functional significance are discussed.     

The structural relationships between 54,000-molecular-weight cellular tumor antigens detected in viral- and nonviral-transformed cells

SV40-transformed mouse cells and murine embryonal carcinoma cells contain a 54,000 MW cellular tumor antigen which was detected with antibodies from animals bearing SV40-induced tumors (D. I. H. Linzer and A. J. Levine, 1979, Cell, 17, 43–52). An antigen with a similar molecular weight has been detected in a variety of transformed mouse cell lines employing an antisera raised against a chemically induced murine sarcoma (A. B. DeLeo et al., 1979, Proc. Nat. Acad. Sci. USA, 76, 2420–2424). To examine the structural relationships between these cellular tumor antigens, the 54,000 MW proteins from a variety of murine transformed cell lines have been analyzed by chromatography of the methionine-containing tryptic peptides derived from these proteins. The results of this analysis demonstrate: (1) SV40 tumor sera and a monoclonal antibody, directed against a cellular tumor antigen of a chemically transformed cell line, each immuno-precipitated the same 54,000 MW protein from SV40-transformed cells as shown by the fact that each protein had an identical peptide map. (2) The 54,000 MW proteins obtained from (a) murine embryonal carcinoma cells, (b) 3T12 cells, (c) chemically transformed cell lines, and (d) an in vitro translation of m-RNA from SV40-transformed cells, all had similar or identical peptide maps. The 54,000 MW proteins from all these sources had eight methionine-containing peptides in common with the 54,000 MW protein obtained after in vivo labeling of SV40-transformed cells in cell culture. However, the 54,000 MW protein derived from SV40-transformed cells labeled in vivo produced a variable number (3–5) of additional methionine-containing tryptic peptides not detected in the other cell lines or with the 54,000 MW protein translated in vitro. These additional peptides may be the result of a post-translational modification of the 54,000 MW protein that is specific to SV40-transformed cells. The results of these experiments demonstrate the structural similarity or identity of the cellular 54,000 MW tumor antigens derived from a variety of murine cell lines transformed by diverse agents.     

Quantitative analysis of cell surface-associated SV40 large t antigen using a newly developed H-protein a binding assay

We have established a sensitive assay for the quantitative determination of large T antigen determinants on the surface of living simian virus 40 (SV40)-transformed cells (mKSA). Cells in suspension culture were incubated with monoclonal antibodies specific for large T antigen (KT3, directed against the carboxyterminus of large T antigen, and PAb 108, directed against an aminoterminal determinant on large T antigen). After incubation with secondary antibody (rabbit anti-mouse IgG), followed by incubation with ³H-protein A, the cells were sequentially extracted first with the nonionic detergent NP-40, followed by ultrasonication and extraction with the zwitterionic detergent Empigen BB. NP-40 solubilized large T antigen associated with NP-40-soluble constituents of the plasma membrane, whereas Empigen BB solubilized the plasma membrane lamina-associated subclass of large T antigen (U. Klockmann and W. Deppert, 1983, EMBO J., 7, 1151–1157). The amount of cell surface-bound ³H-protein A in the NP-40 and Empigen BB extracts was determined by liquid scintillation counting. In agreement with earlier reports, cell surface large T antigen was mainly found in association with the plasma membrane lamina (PML). Since the specific activity of ³H-protein A was known, it was possible to calculate the number of surface-bound ³H-protein A molecules, and thus to estimate the average number of surface-exposed amino- and carboxyterminal determinants of large T antigen per cell. KT3 recognized about 450–900 carboxyterminal determinants, while PAb 108 bound to about 1200–2400 aminoterminal determinants on the surface of a single mKSA cell. The cellular protein p53 also was detected on the surface of mKSA cells and was found to be present in amounts comparable to cell surface large T antigen.     

Association of SV40 large tumor antigen and cellular proteins on the surface of SV40-transformed mouse cells

A cellular protein with a molecular weight of about 53,000 (53K) and histocompatibility antigens (mouse H-2 antigens) have been reported to be associated with viral-specified proteins in transformed cells. We investigated whether such associations could be detected on the surface of SV40-transformed mouse cells. A differential immunoprecipitation technique was adapted so that surface-associated antigens could be detected independently from intracellular antigens. Cells grown as monolayers were enzymatically labeled with ¹²⁵I-Na using a lactoperoxidase-catalyzed reaction, or metabolically labeled with either [³⁵S]methionine or ³²P_i, and were then incubated with antisera against mouse H-2 antigens or SV40 large T-antigen (T-ag) or with monoclonal antibodies against mouse 53K nonviral T-antigen (nvT-ag). The cells were then disrupted with an NP40 solution, the extracts were clarified by centrifugation, and the immune complexes in the supernatant fluids adsorbed with protein A-containing Staphylococcus aureus. Internal antigens, present in the cell lysates, were precipitated by a second incubation with antiserum and the antigen-antibody complexes collected again with immunoadsorbent. The precipitated proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. Reconstruction experiments established that T-ag released from the nucleus during the extraction procedure was not combining with free antigen-binding sites on antibodies bound to the cell surface in the external reaction, that nuclear unbound T-ag was not exchanging with bound surface antigen during extraction, and that the surface reaction was not due to nuclear T-ag released from dead cells and nonspecifically adsorbed onto the surface of living cells. Iodinated 94K T-ag was specifically immunoprecipitated by T antibody during the external reaction; an iodinated 53K polypeptide was coprecipitated. Conversely, labeled T-ag and 53K were coprecipitated from surface-iodinated transformed cells by monoclonal antibodies against mouse 53K nvT-ag. Thus, it appears that SV40 large T-ag and cellular 53K protein are associated on the surface as well as within SV40-transformed mouse cells. In contrast, no detergent-stable complex between T-ag and mouse H-2 antigens was detected on the transformed cells. The possibility that molecular interactions between viral- and cell-coded proteins could be involved in determining some of the observed transformation-related cellular phenotypic changes is discussed.     

Induction of simian virus 40 transplantation immunity in hamsters by gel-purified SV40 large T antigen

Simian virus 40 (SV40) large T antigen and p53 cellular protein were isolated from an SV40-transformed hamster cell line by immunoprecipitation with anti-T sera and purified by sodium dodecyl sulfate-gel electrophoresis. These two protein were tested in hamsters for the presence of SV40 transplantation rejection antigenic sites by in vivo transplantation rejection assay. The large T antigen immunized the hamsters against a challenge of SV40 tumor cells and the protected animals generated cytotoxic spleen cells. Hamsters immunized with the p53 cellular protein were not protected against SV40-induced tumor but there was some delay in the appearance of tumor.     

Properties of simian virus 40 and BK virus tumor antigens form productively infected and transformed cells.

Simian virus 40 (SV40)-specific tumor antigen (T Ag) was immunoprecipitated from extracts of productively infected cells and virus-transformed cells and had the same molecular weight of approximately 97,000 as measured by acrylamide gel electrophoresis. In addition to T Ag proteins of that size, lower molecular weight forms were immunoprecipitated with anti-T serum. Experiments using the protease inhibitor l-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK) indicated that nearly all of the smaller SV40 T Ag polypeptides were artifacts of the extraction procedure. The BK virus-specific T Ag was immunoprecipitated from extracts of infected human cells and coelectrophoresed with SV40 T Ag (97,000 daltons).