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.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg). VI. Mechanism of induction of SV40 transplantation immunity in mice by purified SV40 T antigen (D2 protein)

According to the base sequence homology of the gene coding for the nonstructural (NS) protein the influenza A viruses can be divided into at least three groups. Within each group the homology is about 90% or higher. The avian influenza A viruses fall at least into two groups between which the homology is about 40%. All human strains tested belong to another group. Influenza viruses of other species might comprise their own group(s). The related regions of the NS gene among the two groups of avian influenza viruses overlap completely and they are highly conserved. The results are discussed in terms of a selection pressure concerning the function exerted by the host during the evolution of the different NS genes, which also might explain a certain species specificity concerning the NS gene of influenza A viruses.     

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). VII. Induction of SV40 TrAg in nonpermissive mouse cells by early viable SV40 deletion (o.54/0.59) mutants

Early viable deletion (0.54/0.59) mutants were tested for their ability to induce SV40 transplantation antigen (TrAg) in infected or transformed nonpermissive mouse cells in an attempt to determine the requirement for small t antigen in the expression of TrAg at the cell surface. The results indicate that dl (0.54/0.59) mutants are as efficient as wild-type SV40 in generating specific cytotoxic lymphocytes in C57BU6 mice and in immunizing BALB/c mice against an SV40 tumor cell challenge. Mouse (C57BU6) cells transformed by these mutants were also susceptible to lysis by the specifically sensitized lymphocytes. It can, therefore, be concluded that the synthesis of small t antigen is not an absolute requirement for expression of SV40 TrAg in SV40-infected or -transformed nonpermissive 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.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) II. Isolation and characterization of additional temperature-sensitive mutants of SV40.

Fourteen independent temperature-sensitive mutants of simian virus (SV40) were isolated following nitrous acid or hydroxylamine mutagenesis. Three mutants were assigned to the A group and seven to the BC group on the basis of standard qualitative and quantitative complementation assays. Three other mutants did not complement mutants of any complementation group well under standard conditions nor was delayed complementation observed in quantitative assays. However, these mutants were shown to complement members of the A and BC complementation groups but not members of the D group when the qualitative complementation test was modified by allowing the parental virions to uncoat at permissive temperature prior to incubation at 41°. The assignment of these mutants to the D group was substantiated by demonstrating the wild-type infectivity of DNA extracted from cells infected at 33° for growth at 41°. Thirteen of the mutants were tested for the production of tumor (T), capsid (C), virion (V), and major coat protein (VP1) antigens at permissive and nonpermissive temperature by immunofluorescence assays along with mutants which have been described previously by others for comparison. The temperature-sensitive (ts) mutants isolated in this study produced fully immunoreactive T antigen at both temperatures. None of the tsA mutants produced C, VPl, or V antigens at elevated temperature. The BC mutants isolated in this study all produced T antigen at 41°. These late mutants demonstrated two patterns of expression of virion antigens. One group synthesized C, V, and VP1 at 41° and were indistinguishable from wild type on the basis of antigenic phenotype. A second group showed cytoplasmic and nucleolar fluorescence for C and VPl antigens at the nonpermissive temperature similar to that observed for tsBCll previously. Mutants in this group did not produce V antigen at high temperature.     

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.     

Phosphorylation of SV40 large T antigen in SV40 nucleoprotein complexes

Plaques produced by our P^? mutants of vesicular stomatitis virus (VSV), which are defective in the inhibition of total protein synthesis in infected cells, stop increasing in size after several days of incubation under conditions where those produced by P⁺ mutants increase linearly in size. The basis for the arrest in size of P^? plaques has been shown to be due to the induction of interferon, and the phenotype is termed PIF⁺ for “plaque interferon positive.” Thus P^? plaques can inhibit the increase in size of adjacent P⁺ plaques and the factor responsible has the biological and physical properties of interferon. Also P^? mutants, when plaqued on VERO cells which cannot be induced to produce interferon, produce plaques which increase linearly in size like P⁺ plaques. Finally, the inclusion of anti-interferon antibody in the overlay medium also causes P^? mutants to produce plaques like P⁺ mutants. VERO cells were found to be useful to separate the effects of is mutations on plaque size from the interferon effect. Using other cell types the latter effect (PIF assay) can be used as an assay for the ability of viruses to induce interferon, for the isolation of PIF⁺ mutants from PIF^? virus, and as a test for the ability of cells to respond to interferon induction by PIF⁺ viruses. The assay can be increased in sensitivity through the use of specific cell types and of cell cultures preincubated for several days in the stationary phase of growth. In its most sensitive form, the assay could detect PIF⁺ behavior in certain ts mutants of VSV at permissive temperatures and in VSV mutants emerging from persistent infection. The assay has also been used to isolate novel mutants of VSV which show alterations in the viral P function.     

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.     

Antiserum to polyacrylamide gel-purified simian virus 40 T antigen.

Antisera used previously to assay SV40 T antigen have been produced in tumor-bearing hamsters. This report describes the production and characterization of antiserum produced by injection of rabbits with denatured large-T antigen (Ag) purified by immunoprecipitation and electrophoresis. The resulting antiserum reacts with native large-T Ag as determined by indirect immunofluorescence, complement fixation, and indirect immunoprecipitation assays and binds to species such as “small-T Ag” and previously described proteolytic fragments of large-T Ag.     

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.     

Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines

Large tumor antigen (T antigen) occurs in at least three different oligomeric subclasses in cells infected or transformed by simian virus 40 (SV40): 5-7 S, 14-16 S, and 23-25 S. The 23-25 S form is complexed with a host phosphoprotein (p53). The DNA binding properties of these three subclasses of T antigen from nine different cell lines and free p53 protein were compared using an immunoprecipitation assay. All three subclasses of T antigen bound specifically to SV40 DNA sequences near the origin of replication. However, the DNA binding activity varied between different cell lines over a 40- to 50-fold range. The 23-25 S and 14-16 S forms from most of the cell lines tested bound much less SV40 origin DNA than 5-7 S T antigen. The free p53 phosphoprotein did not bind specifically to any SV40 DNA sequences.     

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.     

Immunological cross-reaction between large T-antigens of SV40 and polyoma virus

Serum containing a high titer of antibodies against SV40 T-antigen was produced in rabbits, using D2 protein as immunogen. This protein was purified from cells infected with the adenovirus-SV40 hybrid virus Ad2⁺D2. Anti-D2 serum recognized SV40 large T, but not small T. In addition, it was able to immunoprecipitate polyoma virus large T, demonstrating that these proteins share antigenic determinants. The titer of this antiserum against polyoma virus large T was comparable to the titers of polyoma virus antitumor sera. Anti-D2 serum did not immunoprecipitate polyoma virus middle T or small T antigens. The activity against polyoma virus large T was sensitive to SDS denaturation.     

Cross-reaction of BK virus large T antigen with monoclonal antibodies directed against SV40 large T antigen

Seventy-five percent of the amino acid sequence of simian virus 40 (SV40) large T antigen is identical to that of the large T antigen of the human papovavirus, BK virus (BKV). Cross-reactivity between BKV T antigen and monoclonal antibodies directed against SV40 T antigen was studied by immunofluorescence, an enzyme immunoassay, immunoprecipitation of radiolabeled extracts, and Western blotting. BKV T antigen was found to be recognized by two monoclonal antibodies, PAb 416 and 430, which react with two distinct sites toward the amino terminus of SV40 large T antigen. These two sites may correspond to two hydrophilic regions of shared amino acid sequence which exist toward the amino termini of the T antigens.     

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.     

Tumorigenicity of SV40-transformed human and monkey cells in immunodeficient mice

Human cells transformed in vitro by SV40 to the anchorage-independent state rarely form tumors in nude mice and therefore constitute an important exception to the otherwise tight correlation between anchorage independence and cellular tumorigenicity. In this paper we explore a number of possible explanations for this unusual situation. We find that the phenomenon is not restricted to human cells but includes monkey cells as well. The nontumorigenic phenotype of the primate SV40 transformants is highly stable. We are unable, through selection of ever more anchorage-independent lines, to generate a primate SV40 transformant which will grow as a tumor in even the most immunologically crippled animals. One tumor was obtained from SV80 (an SV40-transformed human cell line) following injection into a mouse deficient in both T and B cell functions. However, cell lines derived from this tumor are not significantly more tumorigenic than the SV80 parent. This low incidence of tumor formation is not due to the fact that the primate cells are transformed by nononcogenic defective viral genomes nor to a nutritional inadequacy of the host animal for the growth of human cells. Although a T cell-independent mechanism may be the major mechanism involved in tumor suppression, it is unlikely that this completely accounts for the general lack of tumor growth by most of these cells. It appears that the interaction of SV40 (a primate virus) with primate cells may be intrinsically less oncogenic than its interaction with rodent cells.     

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.     

Effect of nuclear localization of large tumor antigen on growth potential of SV40-transformed cells

Transport-defective mutants of PARA, an SV40-adenovirus 7 hybrid virus, induce the synthesis of SV40 large T-antigen (T-ag) that is not transported to the nucleus and accumulates in the cytoplasm (cT-ag) of infected and transformed cells. The ability of cT mutants of PARA to induce phenotypic transformation of hamster embryo fibroblasts was examined. The cytoplasmic localization of T-ag was not stable, as extensive subculturing resulted in cell populations comprised of mixtures of cells which expressed T-ag in either the nucleus and/or the cytoplasm. Clonal analysis resulted in the selection of two cell populations: one cell type expressed T-ag solely in the cytoplasm and the second contained a mixture of cells which expressed T-ag in the nucleus and/or the cytoplasm. Serial subculture of clonal cell lines containing T-ag exclusively in the cytoplasm resulted again in the evolution of mixed populations which expressed T-ag in the nucleus and/or the cytoplasm. Analysis of synchronized cultures of such mixed clonal lines revealed that the intracellular distribution of T-ag was under the influence of the cell cycle; T-ag was present in the nucleus during S-phase, the period of maximal DNA synthesis, and in the cytoplasm during other phases of the cell cycle. These results suggest that a selective advantage exists during in vitro culturing for cells with a partial reversion of the cT phenotype. Examination of the growth properties of clonal lines in vitro indicated that cells in which T-ag could be detected in the cytoplasm, either continuously or during certain phases of the cell cycle, exhibited reduced growth potential under stringent culture conditions relative to wild-type transformants, behaving more like minimal transformants. In addition, the capacity for tumor induction in weanling hamsters by PARA(cT)-transformed hamster cells was reduced substantially in comparison to wild-type transformed cells. These results suggest that the constant presence of SV40 T-ag in the nucleus promotes maximal expression of the transformed cell phenotype.