Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus.

The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.     

Structural prerequisites of simian virus 40 large T antigen for the maintenance of cell transformation.

We have investigated the functional roles of two structural subsets of simian virus 40 (SV40) large T antigen, namely homo-oligomers and complexes with the host cellular p53 protein, for the transformed phenotype. We examined T antigen produced in cells transformed by temperature-sensitive SV40 large T mutants: heat-sensitive or unrestricted SV40 tsA58-transformed rat cells and unrestricted tsA1499 transformants. In both unrestricted cell lines, T antigen was temperature-sensitive only for the formation of fast sedimenting homo-oligomers. Corresponding to our recent observations obtained with tsA1499-infected monkey cells, in tsA1499 transformants large T was competent to form stable T-p53 complexes independently of the temperature. However, T antigen coded for by tsA58, which is heat-sensitive for binding to p53, occurred in stable complexes with this protein in unrestricted tsA58 transformants under all conditions. Furthermore, in both unrestricted transformants T-p53 complexes arise in the absence of homo-oligomers of T antigen. In conclusion, T antigen homo-oligomers are not involved in cell transformation, whereas T-p53 complexes may be involved in the maintenance of this phenotype.     

Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension.

A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system.     

Polyoma virus middle t antigen: a tumor progression factor.

Polyoma virus (PyV) deletion mutant dl23 (affecting both large T and middle t but not small t antigens) was used to study transformation of 3T3 rat cells. This mutant generated stable transformants in the agar assay at a frequency similar to that of wild-type virus (WT). However, WT-induced transformants were detected 3 weeks after infection, whereas those induced by the mutant could not be detected until 6 to 8 weeks after infection. In this respect, dl23 PyV behaved like WT simian virus 40 (SV40). Cells transformed by WT SV40 or by dl23 PyV were similar in all their transformed properties. Those transformed by WT PyV were different from the others on the basis of morphology, cell adhesion to the substrate, release of protease activity, efficiency of doubling in agar, growth rate, and time required for tumor formation. Saturation density, the ability to grow in agar, the serum requirement for cloning, and the ability to grow on a cell monolayer were similar for all transformants. Middle t antigen enhanced membrane alterations and growth rate of the transformed cells, shortening the time required for tumor formation in rats.     

A fragment of the simian virus 40 early genome can induce tumors in nude mice.

Cell lines transformed by simian virus 40 mutant F8dl (deleted from 0.168 to 0.424 map units, corresponding to the carboxy-terminal 62% of the wild-type simian virus 40 large tumor antigen) are tumorigenic in nude mice. Four of five C3H10T1/2 cell lines transformed by F8dl were tumorigenic in nude mice, whereas two of two wild-type transformants were tumorigenic.     

Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.

To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.     

Characterization of am404, an amber mutation in the simian virus 40 T antigen gene.

We analyzed the biological activity of an amber mutation, am404, at map position 0.27 in the T antigen gene of simian virus 40. Immunoprecipitation of extracts from am404-infected cells demonstrated the presence of an amber protein fragment (am T antigen) of the expected molecular weight (67,000). Differential immunoprecipitation with monoclonal antibody demonstrated that am T antigen was missing the carboxy-terminal antigenic determinants. The amber mutant was shown to be defective for most of the functions associated with wild-type T antigen. The mutant did not replicate autonomously, but this defect could be complemented by a helper virus (D. R. Rawlins and N. Muzyczka, J. Virol. 36:611-616, 1980). The mutant failed to transform nonpermissive rodent cells and did not relieve the host range restriction of adenovirus 2 in monkey cells. However, stimulation of host cell DNA, whose functional region domain has been mapped within that portion of the protein synthesized by the mutant, could be demonstrated in am404-infected cells. A number of unexpected observations were made. First, the am T antigen was produced in unusually large amounts in a simian virus 40-transformed monkey cell line (COS-1), but overproduction was not seen in nontransformed monkey cells regardless of whether or not a helper virus was present. This feature of the mutant was presumably the result of the inability of am T antigen to autoregulate, the level of wild-type T antigen in COS-1 cells, and the unusually short half-life of am T antigen in vivo. Pulse-chase experiments indicated that am T antigen had an intracellular half-life of approximately 10 min. In addition, although the am T antigen retained the major phosphorylation site found in simian virus 40 T antigen, it was not phosphorylated. Thus, phosphorylation of simian virus 40 T antigen is not required for the stimulation of host cell DNA synthesis. Finally, fusion of am404-infected monkey cells with Escherichia coli protoplasts containing appropriate procaryotic suppressor tRNAs showed that am404 is a suppressible nonsense mutation.     

Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40.

Chinese hamster lung (CHL) cells transformed by wild-type simian virus 40 (cell line CHLWT15) or transformed by the simian virus 40 mutants tsA30 (cell lines CHLA30L1 and CHLA30L2) or tsA239 (cell line CHLA239L1) were used to determine the rates of turnover and synthesis of the T-antigen protein and the rate of turnover of the phosphate group(s) attached to the T-antigen at both the permissive and restrictive temperatures. The phosphate group turned over several times within the lifetime of the protein to which it was attached, with the exception of the phosphate group in the tsA transformants at 40 degrees C, which turned over at the same rate as the T-antigen protein. The steady-state levels of the T-antigens (molecular weights, 92,000 [92K] and 17K) and the amount of simian virus 40-specific RNA was also determined in each of the lines. The CHLA30L1 line contained two to three times more early simian virus 40 RNA than the CHLA30L2 line; although neither line formed colonies in agar at 40 degrees C, CHLA30L1 overgrew a normal monolayer at 40 degrees C. The rate of 92K-T-antigen synthesis was 1.5 times faster in CHLA30L1 than in CHLA30L2 at 33 degrees C and 4 times faster at 40 degrees C. The different phenotype of these two presumably isogenic cell lines seem to be related to the levels of the T-antigens. The ratios of the 92K T-antigen to the 17K T-antigens were similar in the two lines. Transformed CHL cell lines, unlike transformed mouse 3T3 cell lines, were found to contain very small amounts of the 56K T-antigen.     

A functional simian virus 40 origin of replication is required for the generation of a super T antigen with a molecular weight of 100,000 in transformed mouse cells.

We used two recombinant plasmids, one containing wild-type simian virus 40 DNA (pSVR1) and the other containing a simian virus 40 genome with a defective origin of replication (pSVR1-origin-minus) to transfect NIH3T3 cells. Quantitation of T-antigen synthesis by indirect immunofluorescence at 48 h after transfection with either DNA revealed the same percentage of T-positive nuclei. The transformation frequencies observed were also similar with both plasmids. Immunoprecipitation of [35S]methionine-labeled cell extracts showed the expected 94,000-dalton (94K) T and 17K t antigens in all clones examined. In pSVR1-generated transformants, a 100K super T antigen was also detected. Transformants isolated from pSVR1-origin-minus transfection, however, never expressed this 100K super T antigen, and some of these clones originally also showed greatly reduced levels of 94K T antigen. However, after growth in culture for several generations, the levels of 94K T antigen synthesis in these underproducer clones were dramatically increased. A direct correlation between the amounts of T antigen synthesized and the ability to grow independently of anchorage was observed. The mechanism which brings about increasing levels of T-antigen synthesis in some of the clones is not clear, but it appears not to be due to changes in either the copy number or the methylation pattern of the integrated simian virus 40 DNA.     

Effect of Ultraviolet Irradiation on the Survival of Simian Virus 40 Functions in Human and Mouse Cells

The relative sensitivities to ultraviolet light of various simian virus 40 (SV40) functions were studied in human and mouse cells. Transformation appeared to be less ultraviolet (UV)-sensitive than either T or V antigen when all functions were compared in the same cell. However, the time course of both T- and V-antigen appearance was delayed with UV-irradiated virus, so that the survival curves of these functions changed with time. Mouse and human cells which were transformed by UV-SV40 all contained SV40 T antigen. Infectious virus could be recovered from many more transformants than would be expected from the infectivity in African green monkey kidney cells of the irradiated virus. The results suggest that human and mouse cells are capable of reactivating UV-damaged SV40.     

Rat cells transformed by simian virus 40 give rise to tumor cells which contain no viral proteins and often no viral DNA.

Rat 3T3 cells transformed by simian virus 40 were injected into rats to examine their capacity to develop into tumors. Both large T-dependent (N) transformants and large T-independent (A) transformants were used. All the transformed cell lines contained large T and small t and could multiply efficiently in agar. Only some transformants could develop into tumors. All tumor cells examined had lost both large T and small t. Tumor cells in which the viral genome could still be detected were found together with tumor cells in which the simian virus 40 DNA could no longer be detected. N transformants which displayed the transformed phenotype in a temperature-sensitive manner became temperature insensitive during tumor formation.     

Murine cell complementation of a cold-sensitive defect for simian virus 40 replication in simian cells.

Mouse 3T3 fibroblasts were found to complement, in simian cell variants semipermissive to simian virus 40, a cold-sensitive defect of an early function, but not a nonconditional defect of viral uncoating. The variant simian cells could rescue simian virus 40 from 3T3 transformants, and this capacity was not temperature dependent.     

Use of simian virus 40 replication to amplify Epstein-Barr virus shuttle vectors in human cells.

We have increased the copy number of Epstein-Barr virus vectors that also carry the origin of replication of simian virus 40 (SV40) by providing a transient dose of SV40 T antigen. T antigen was supplied in trans by transfection of a nonreplicating plasmid which expresses T antigen into cells carrying Epstein-Barr virus-SV40 vectors. A significant increase in vector copy number occurred over the next few days. We also observed a high frequency of intramolecular recombination when the vector carried a repeat segment in direct orientation, but not when the repeat was in inverted orientation or absent. Furthermore, by following the mutation frequency for a marker on the vector after induction of SV40 replication, it was determined that SV40 replication generates a detectable increase in the deletion frequency but no measurable increase in the frequency of point mutations.     

Growth control in simian virus 40-transformed rat cells: temperature-independent expression of the transformed phenotype in tsA transformants derived by agar selection.

Fisher rat fibroblasts (FR 3T3), transformed with the tsA30 mutant of simian virus 40 and selected by colony formation in soft agar, maintained the transformed phenotype at high temperature, whereas most transformants isolated from foci were found to undergo a phenotypic reversion toward the normal state in their saturation density, ability to grow in soft agar, and rate of 2-deoxyglucose transport. The temperature-independent phenotype observed in agar-selected transformants was not due to a reversion of the viral mutation. These results, similar to those previously obtained with polyoma virus tsa mutants, further suggest that two distinct mechanisms may operate in both cases for maintaining the transformed phenotype. Immunofluorescence studies suggested a different regulation of T antigen synthesis in these two classes of transformants.     

Distinct transformation phenotypes induced by polyoma virus and simian virus 40 in rat fibroblasts and their control by an early viral gene function.

Several transformed cell lines established from Fisher rat cells (FR 3T3) infected with wild-type polyoma virus or simian virus 40 or early temperature-sensitive mutants (polyoma tsa and simian virus 40 tsA30) were studied for their transformation phenotypes. The distinct patterns which were obtained for polyoma and simian virus 40 transformants led to the conclusion that these two viruses express different transforming abilities in rat cells. The results obtained with temperature-sensitive mutant-derived transformants indicate that all of the transformation characteristics studied so far may be under the control of a viral function in polyoma tsa-transformed cells.     

Retinoblastoma protein and simian virus 40-dependent immortalization of human fibroblasts.

Transformation and immortalization of human diploid fibroblasts by simian virus 40 (SV40) is at least a two-stage process, since transformants have a limited lifespan in culture. We have isolated immortalized derivatives (AR5 and HAL) from transformants generated with an origin-defective SV40 genome encoding a heat-labile large T protein (T antigen) and reported that both preimmortal and immortal transformants are continuously dependent on T antigen function for growth as determined by temperature shift experiments. In this study, we demonstrate complex formation between T antigen and the retinoblastoma susceptibility gene product (Rb) at 35 degrees C and observed a reduction in complexes under conditions of loss of T antigen function and growth inhibition at 39 degrees C. Viral oncogenes (polyomavirus large T protein and adenovirus E1A 12S protein) known to bind Rb were introduced into AR5 and HAL cells, both stably by gene transfer and transiently by virus vectors. Such double transformants are still unable to proliferate at 39 degrees C, although complex formation with the newly introduced oncogenes was demonstrated. We suggest that T antigen interacts with other cellular processes in addition to Rb to transform and immortalize human cells in culture. Our finding that p53-T antigen complexes are also temperature dependent in AR5 and HAL cells could provide such an additional mechanism.     

Temperature dependency for maintenance of transformation in mouse cells transformed by simian virus 40 tsA mutants.

Mouse embryo fibroblasts and 3T3 cells were transformed by wild-type, tsB4, tsA7, tsA58, and tsA209 simian virus 40. Clones of transformants were generated both in soft agar and in liquid medium by focus formation and at both high and relatively low multiplicities of infection. All transformants were assayed for three phenotypes of transformation: (i) the ability to form highly multinucleated cells in cytochalasin B-supplemented medium, i.e., uncontrolled nuclear division; (ii) the capacity to continue DNA synthesis at increasing cell density; and (iii) the ability to form colonies in soft agar. The great majority of mouse embryo fibroblast transformants generated with tsA mutant virus were temperature sensitive for transformation in all three assays, regardless of the input multiplicity or whether they were generated in liquid medium or soft agar. These transformants exhibited a normal or near-normal phenotype at the nonpermissive temperature of 40 degrees C. All but one of the transformants which appeared transformed at both temperatures were in the A209 group. In contrast to mouse embryo fibroblasts, transformants generated with 3T3 cells and tsA virus were often not temperature sensitive, exhibiting the transformation phenotypes at both temperatures. This phenomenon was more often observed when 3T3 transformants were generated in soft agar. These results, along with other published data, suggest that uncontrolled nuclear division and uncontrolled DNA synthesis are a function of the simian virus 40 A gene. Finally, with the 3T3 transformants, there was often discordance in the expression of transformation among the three phenotypes. Some tsA transformants were temperature sensitive in one of two assays but were transformed at both 33 and 40 degrees C in the remaining assay(s). Other transformants exhibited a normal cytochalasin B response at either temperature but were temperature sensitive in the other assays.     

Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts.

Recombinant retroviruses that transduce the simian virus 40 (SV40) large T antigen or the polyomavirus large T antigen as well as encoding resistance to antibiotic G418 were used to investigate whether these genes alone were sufficient for immortalization of primary cells. The results provided definitive evidence that either viral gene can efficiently establish primary fibroblasts. The capability of the SV40 large T antigen to establish primary fibroblasts was undiminished by a mutation that alters its binding to sequences within the origin of replication. Surprisingly, most of the primary cells established by the expression of the SV40 large T antigen did not have a transformed phenotype. This suggests that transformation by SV40 is not simply due to a high level of expression of the SV40 large T antigen and stabilization of cellular p53.     

Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity.     

Simian virus 40 gene A function and maintenance of transformation.

Transformants have been isolated after infection of rat embryo cells at 33 C with either wild-type simian virus 40 or with the temperature-sensitive gene A mutants, tsA7 and tsA28. Examination of properties usually associated with transformation such as growth in 1% serum, growth rate, saturation density, and morphology show that these properties are temperature dependent in the tsA transformants characterized, but are not temperature dependent in the wild-type transformants that have been examined. In the most thoroughly characterized tsA transformants the expression of T antigen also appears to be temperature dependent. These data suggest that an active A function is required for the maintenance of transformation in these cells. In the lytic cycle, the A function is involved in the initiation of DNA synthesis. Thus transformation by simian virus 40 may be the direct consequence of the introduction of the simian virus 40 replicon and the presence of its DNA initiator function, which causes the cell to express a transformed phenotype.