Characterization of murine thymic stromal-cell lines immortalized by temperature-sensitive simian virus 40 large T or adenovirus 5 E1a.

The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33 degrees C and arrest growth when transferred to the nonpermissive temperature of 39 degrees C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined.     

Variant lines of mouse kidney cells transformed by an SV40tsA mutant with growth properties of wild-type transformed cells at nonpermissive temperature.

MKSA207 cells, a BALB/c mouse kidney line transformed by a tsA mutant of SV40, are temperature-dependent for the expression of the 'standard transformed phenotype'. At the permissive temperature (33.5 degrees C), the mKSA207 cells resembled wild-type (wt) SV40 transformants; they contained the intranuclear SV40 T antigen, grew to high saturation density in monolayer culture in either 10% or 0.5% serum, and also in methylcellulose suspension culture and became multinucleate in cytochalasin B. At the nonpermissive temperature (39.8 degrees C), the mKSA207 cells lost some of their transformed properties; they grew only to low density in 10% serum, hardly grew at all in 0.5% serum or in methylcellulose suspension culture, and remained mono- or binucleate in cytochalasin B. At 40 degrees C in low serum, mKSA207 cells lost the intranuclear T antigen and when fed 10% serum at 39.8 degrees C, accumulated large amounts of T antigen in the cytoplasm. Derivatives of mKSA207 have been selected at 39.8 degrees C in liquid medium and methylcellulose suspension culture. The heat adapted lines, like wt SV40 transformants, exhibited the standard transformed phenotype at both 33.5 and 39.8 degrees C. It is unlikely that acquisition of temperature-independence for the transformed phenotype was due to reversion of the tsA gene to wild-type because the heat-adapted cell lines displayed the cytoplasmic T antigen at 39.8 degrees C, characteristic of the parental mKSA207 cells and SV40 rescued from one of the heat-adapted lines was temperature sensitive for growth. The T antigen levels (complement fixation units per 10(6) cells) of heat-adapted lines grown at 39.8 degrees C were comparable to those of mKSA207 cells grown at 33.5 or 39.8 degrees C.     

Highly efficient establishment of immortalized cells from adult human liver

Human liver tissue slices, homogenates, organ cultures, subcellular fractions and primary hepatocyte cultures represent valuable tools for analysis of metabolism, toxicity and efficacy of xenobiotics. However, the limited access to human tissue and the high test variability with different tissue isolates underline the significance of having standardized and defined human liver cell models. This report describes the establishment of replicative cultures of human liver epithelial cells and their immortalization in serum-free medium. Highly efficient immortalization is achieved by the infection of primary liver cells with a retroviral recombinant carrying the simian virus 40 (SV40) large T antigen gene. These cultures have an indefinite life-span, are non-tumorigenic and express differentiation markers and metabolic functions of normal primary hepatocytes. Therefore, these cells are useful for various pharmaco-toxicological applications.Abbreviations LCM live cell medium - SV40 Simian virus 40     

Characterization of Murine Thymic Stromal-Cell Lines Immortalized by Temperature-Sensitive Simian Virus 40 Large T or Adenovirus 5 E1a

The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33°C and arrest growth when transferred to the nonpermissive temperature of 39°C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined.     

Studies on the regulation of deoxypyrimidine kinases in normal, SV40-transformed and SV40- and adenovirus-infected mouse cells in culture.

Deoxythymidine kinase, deoxyuridine kinase and deoxycytidine kinase were each found to be present in multiple enzymatic forms in mouse cells. Five different criteria were employed to distinguish between these multiple enzyme activities: 1) electrophoretic mobility in polyacrylamide gels, 2) substrate specificity, 3) sensitivity to parahydroxymercurybenzoate, 4) the ability of different ribonucleoside triphosphates to act as phosphate donors, 5) feedback inhibition of enzyme activity by deoxyribonucleoside triphosphates.Actively growing 3T3 cells in culture expressed a substrate-specific deoxycytidine kinase and a deoxythymidine-deoxyuridine kinase activity. Resting cells in culture contained two electrophoretically distinct deoxypyrimidine kinase activities that phosphorylated all three deoxypyrimidine nucleoside substrates. The same kinases detected in actively growing cells in culture were the major enzymatic activities observed in fetal or young mice, while adult mice expressed predominantly those activities found in resting cells in tissue culture. Thus the expression of the mouse deoxypyrimidine kinases reflects alterations in cell growth, indicating that their regulation is governed by the physiological state of the cells.SV-40-transformed derivatives of three different mouse cell lines displayed a qualitatively normal mode of deoxypyrimidine kinase regulation. Growing transformed cells expressed the growing form of these enzymes while resting transformed cells at very high saturation densities contained the normal resting cell kinases. When normal resting 3T3 cells were abortively infected with SV40 or adenovirus, there was a large increase in the specific activity of only the growing cell specific kinases.     

Human epithelial cells immortalized by SV40 retain differentiation capabilities in an in vitro raft system and maintain viral DNA extrachromosomally.

Carcinomas are the predominant type of cancer found in man, yet in vitro studies on the transformation of epithelial cells have been limited. In an attempt to extend our knowledge of the mechanisms involved in the development of epithelial cancers, we have examined the effects of oncogenes on keratinocytes in vitro using both the ability to immortalize and the ability to alter differentiation as criteria for transformation. SV40 T-antigen was observed to be an efficient immortalizing agent in human keratinocytes consistent with previous studies in other human cell types. Using an in vitro cell culture system (rafts) for epithelial stratification at the air-liquid interface, we observed that the morphology of rafts of SV40-immortalized keratinocytes was similar to that of untransfected epithelial cells, demonstrating that although immortal these cells retain differentiation capabilities. The ability to differentiate was lost only upon prolonged passage in culture, suggesting that this effect is separable from immortalization. In these immortalized epithelial cells, SV40 genomes were found to be maintained as a heterogeneous population of extrachromosomal molecules dependent upon the SV40 origin of replication. It is not clear whether these molecules arise continuously as a result of excision events from integrated copies or are stably maintained as episomes.     

Androgen-dependent prostate epithelial cell selection by targeting ARR(2)PBneo to the LPB-Tag model of prostate cancer

Cell cultures representing different stages of prostatic carcinoma will be a useful tool allowing a more complete understanding of the role of individual genes in tumorigenesis. We used the androgen-regulated probasin promoter linked to the neomycin phosphotransferase (Neo) gene, to generate the ARR(2)PBneo transgenic mouse model. Development was normal and all six ARR(2)PBneo transgenic founder lines expressed the Neo gene in a prostate-specific manner. Line C, which expressed high levels of neo, was crossbred to LPB-Tag 12T-7f transgenic mice (in which the SV40 large T antigen (Tag) was targeted to the prostate by the large probasin (LPB) promoter). Three bigenic males (carrying both Neo and Tag transgenes) were identified. Prostatic lesions developed in these mice in a predictable and heritable manner, indicating that Neo did not alter Tag-induced prostate tumor development and progression. Three separate NeoTag epithelial cell strains were established from three bigenic mice. G418 selection was used to obtain immortalized epithelial cells in culture. Selected cells expressed the Neo and Tag transgenes, cytokeratins 8 and 18, and were androgen responsive for growth. To determine if these NeoTag cells maintained a similar in vivo phenotype to the 12T-7f transgenic line, tissue recombinations were made with rat urogenital sinus mesenchyme (rUGM) and grafted under the renal capsule of male nude mouse hosts. In recombinants, the three NeoTag strains developed PIN lesions and/or more extensive adenocarcinoma than seen in the 12T-7f mouse. Androgen ablation demonstrated that the grafts were androgen responsive. NeoTag cells grafted without rUGM developed undifferentiated adenocarcinoma demonstrating that prostatic stroma dictates the glandular architecture seen in the well-differentiated adenocarcinoma.     

Conditional transformation of mouse liver epithelial cells. An in vitro model for analysis of genetic events in hepatocarcinogenesis.

Primary rodent and human hepatocytes have a very limited lifespan in culture and are not readily applicable to transformation studies in vitro. To facilitate the investigation of early genetic events involved in hepatocarcinogenesis, we examined a transformation assay system utilizing conditionally immortalized mouse liver epithelial cells as an alternative to primary hepatocytes. By infecting primary mouse hepatocytes with a recombinant retrovirus carrying a temperature-sensitive simian virus 40 large T antigen gene, two mouse liver epithelial cell lines, CHST8 and CHST10-2.1, were established. Because of the heat-labile nature of the large T antigen, the cell lines proliferated rapidly at 33 degrees C, but were growth-arrested at 39 degrees C. Because activated c-H-ras and c-myc oncogenes are frequently found to be involved in mouse hepatocarcinogenesis in vivo, we assessed whether those oncogenes can complement the immortalizing function of the large T antigen at the nonpermissive temperature. When CHST8 cells were doubly transfected with activated c-H-ras and c-myc at 33 degrees C, they exhibited clonal growth ability even after shifting the temperature to 39 degrees C. However, neither c-H-ras nor c-myc alone allowed growth at 39 degrees C. On the other hand, c-H-ras alone was sufficient for overcoming the growth defect of CHST10-2.1 cells at 39 degrees C, whereas c-myc alone was again ineffective. Northern blot studies revealed that endogenous c-myc expression was significantly downregulated in the parental CHST8 cells after a temperature shift from 33 to 39 degrees C. In contrast, in the parental CHST10-2.1 cells, appreciable c-myc expression was observed at both temperatures. These results indicate that c-H-ras and c-myc can cooperate in complementing the ability of the temperature-sensitive large T antigen to immortalize mouse liver cells at the nonpermissive temperature. In addition, the mutant c-H-ras, but not c-myc, cooperated with the functional T antigen at 33 degrees C to allow growth in soft agarose of the CHST8 and CHST10-2.1 cell lines. However, cell lines carrying mutant c-H-ras and overexpressing c-myc were unable to grow in soft agarose at 39 degrees C. Thus, the two cellular oncogenes were insufficient for full transformation of the liver epithelial cells. The present in vitro model should be useful for investigating molecular events involved in both early and late stages of hepatocarcinogenesis.     

Simian virus 40 compensates a cellular mutational defect of a serum-dependent function controlling cell cycle progression in the G2 phase

Rat 3Y1tsF121 fibroblasts are arrested in the G2 phase at the nonpermissive temperature due to a temperature-sensitive (ts) defect, and the G2 arrest is overcome at the nonpermissive temperature by the addition of a large dose of fresh serum. When the G2-arrested cells which had been exposed to the nonpermissive temperature for 12 hr were shifted down to the permissive temperature, most divided within 12 hr. When the cultures prepared in parallel were infected with simian virus 40 (SV40) at the nonpermissive temperature, the G2-arrested cells divided as early as 6 hr after the expression of T antigen. The G2-arrested cells, which had been exposed to the nonpermissive temperature for 36 hr, lost both the ability to restore the G2 and M traverse at the nonpermissive temperature after the addition of fresh serum and the reversibility of the arrest upon shift down to the permissive temperature. However, SV40 induced these cells to divide at the nonpermissive temperature, as in the case of the reversibly arrested cells. A small t-antigen-deletion mutant (dl-884) also induced both types of the G2-arrested cells to divide at the nonpermissive temperature. These results suggest that (1) SV40 compensates or activates, in the G2 phase, the function regulating G2 and M transition by serum; (2) SV40 induces restoration of the irreversible G2 arrest; and (3) small t antigen is not responsible for these activities of SV40.     

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.     

Analysis of host range of nontransforming polyoma virus mutants.

The nontransforming polyoma virus mutant NG-18, screened originally for its ability to grow in polyoma-transformed cells, has now been shown to grow in a variety of other cell types. Among cells found to be substantially permissive for the growth of NG-18 are: Phenotypic revertants of polyoma-transformed 3T3 cells, C-type RNA virus transformed and/or producing 3T3 cells, 3T3 cells that become transformed upon infection with leukemia virus, primary baby mouse kidney epithelial cells, and primary mouse embryo fibroblasts. Among cells found to be poor hosts for the growth of NG-18 are: Several 3T3 cell lines, SV40-transformed 3T3 cells, spontaneously transformed 3T3 cells, radiation- and chemical carcinogen-transformed 3T3 cells, and late passage mouse embryo fibroblasts. Similar results have been obtained using three other independently isolated host range mutants of the same type as NG-18. The permissive state thus does not require a cell-associated polyoma genome as the basis for complementation of growth for this class of host range mutant. In addition, the results demonstrate that cells can be permissive for the growth of such nontransforming virus mutants without themselves possessing properties commonly associated with transformation: Loss of density-dependent inhibition of growth, low serum requirement for growth, loss of anchorage dependence of growth, and lectin agglutinability. Implications of these findings are discussed in reference to possible mechanisms of action of the NG-18 gene in productive infection and transformation.     

Inhibition of measles virus replication by cyclic AMP

In some hybrid cell lines between human and mouse cells the ribosomal RNA (rRNA) genes of both species are present but only the rRNA of the dominant species is expressed. The silent rRNA genes can be reactivated if the hybrid cells are infected with simian virus 40 (SV40). The role of the SV40 early gene, the A gene, in the reactivation of the silent rRNA genes was investigated. A temperature-sensitive mutant of the SV40 early region, tsA58, could not reactivate the silent mouse rRNA genes at the nonpermissive temperature of 39° in human > mouse hybrids, although it could do so at 32°. Wild-type SV40 did induce the expression of mouse rRNA at both 32° and 39°. Adenovirus 2 (Ad2) could not reactivate the silent rRNA genes in these hybrid cells. Infection by either Ad2+D?1 or Ad2+D?2, hybrid adenoviruses which contain portions of the A region of the SV40 genome, led to expression of both species of 28 S rRNA. However, infection by the nondefective adenovirus Ad2+ND-1 (H71), which contains an incomplete SV40 A region, failed to reactivate silent rRNA genes. Hybrid cells infected with the small t deletion mutant of SV40, dl-2005, also showed reactivation. Finally, both human and mouse rRNA were found in SV40-transformed mouse-human hybrid cells and in polyoma virus-infected hybrids. These results indicated that SV40 reactivation of silent rRNA genes in human-mouse hybrids requires the major product of the A gene, the large T antigen.     

SV40 T-antigen-related surface antigen: correlated expression with nuclear T-antigen in cells transformed by an SV40 A-gene mutant

Rat embryo cells transformed by the SV40 A-gene mutant tsA 28.3 were analyzed at permissive and nonpermissive temperatures for the expression of SV40 nuclear T-antigen (T-Ag) and of SV40 T-Ag-related surface antigen (“surface T”) by indirect immunofluorescence microscopy. At permissive temperature both antigens were detected with rabbit antiserum prepared against purified SDS-denatured T-Ag. Upon shift of tsA 28.3 cells to the nonpermissive temperature, expression of both nuclear T-Ag and of surface T decreased simultaneously. When the cells were returned to the permissive temperature, both nuclear T-Ag and surface T reappeared. Analysis of extracts from tsA 28.3 cells grown and labeled with [³⁵S]methionine either at permissive or at nonpermissive temperature by immunoprecipitation and SDS-polyacrylamide gel electrophoresis demonstrated that tsA 28.3 cells synthesized the T-Ag polypeptide only at the permissive temperature. The T-Ag polypeptide was not detected in extracts of cells grown and labeled at nonpermissive temperature. These results demonstrate a strong correlation between the expression of nuclear T-Ag, surface T, and the synthesis of the T-Ag polypeptide, suggesting that both nuclear T-Ag and surface T are products of the SV40 A-gene.     

The cellular chromatin is an important target for SV40 large T antigen in maintaining the transformed phenotype

To identify cellular targets of simian virus 40 large T antigen (SV40 large T) important for the maintenance of cellular transformation, we have compared biological properties of SV40 tsA58 mutant large T antigens expressed in cells of a matched pair of SV40 tsA58 N-type (temperature-sensitive) and A-type (temperature-insensitive) transformants of the normal rat fibroblast line F111 (D. Pintel et al., J. Virol. 38, 518-528, 1981). Characterization of the selected cell lines demonstrated that cells of the N-type transformant [FR(tsA58)A] exhibited properties similar to those of the corresponding SV40 wild-type transformant [FR(wt648)] at the permissive growth temperature (32 degrees ), but reverted to a phenotype indistinguishable from the parental F111 cells at the nonpermissive growth temperature (39 degrees). At both growth temperatures, cells of the A-type transformant [FR(tsA58)57] were very similar to FR(wt648) cells in all properties analyzed. Both mutant-transformed cell lines expressed authentic tsA58 mutant large T antigens at comparable steady-state levels. Analysis of the subnuclear distribution of large T antigens in wild-type and in mutant-transformed cells kept at permissive or at nonpermissive growth temperature, respectively, revealed an important biological difference between the mutant T antigens in N- and A-type transformants: Whereas the subnuclear distribution of wild-type large T in FR(wt648) cells remained unchanged at both growth temperatures, mutant large T in FR(tsA58)A cells (N-type transformant) already 1 day after the shift to the nonpermissive growth temperature no longer stably associated with nuclear substructures, notably the cellular chromatin. In contrast, mutant large T in FR(tsA58)57 cells (A-type transformant) retained this ability. The ability (or inability) of the mutant T antigens to associate with the cellular chromatin in vivo was paralleled by different DNA binding properties of the mutant large T antigens in vitro. Large T in FR(tsA58)A cells no longer bound to the SV40 ORI in vitro after the shift to the nonpermissive growth temperature, whereas large T in FR(tsA58)57 cells at the elevated growth temperature had preserved this activity to a degree similar to its ability to associate with the cellular chromatin. We suggest that in the system of matched pairs of N- and A-type transformants analyzed in this study, expression of the transformed phenotype in FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature is due to the preservation of a biologically active conformation of the mutant large T, allowing it to maintain its interaction with specific targets at the cellular chromatin.     

Rescue of SV 40 from interferon-treated heterokaryons

Summary Simian virus 40 (SV 40)-transformed nonpermissive cells express only the early products of SV 40. Heterokaryons formed by fusion of these transformed cells with uninfected permissive cells support the activation of the resident viral genome leading to subsequent viral DNA replication, late protein synthesis and release of progeny virus. Pretreatment of heterokaryon cultures with either mouse or monkey interferon (IFN) before fusion with polyethylene glycol (PEG) produced a dose-dependent inhibition in the appearance of free viral DNA as well as production of infectious virus. The decreased yield of SV 40 in these cultures was similar to the inhibition which was observed in mouse or monkey cells incubated with homologous IFN prior to exogenous infection with SV 40. when IFN was added to the cultures at progressively later times after fusion with PEG, there was less inhibition of virus production. Although there was a comparable decrease in the production of virus by pretreatment with either mouse or monkey IFN, monkey IFN exerted the inhibition for a longer period of time when added after heterokaryon formation. These results demonstrate that IFN treatment applied even after initiation of SV 40 replication can still inhibit virus multiplication.With 1 Figure     

The effect of interferon on the early functions of simian virus 40.

The effect of interferon (IF) on the early functions of simian virus 40 (SV40) was studied by monitoring the inhibition of SV40-induced T antigen (Ag) in permissive monkey and nonpermissive mouse cells. When IF was added to cells which had previously been transformed by SV40, there was no effect on T Ag production. However, pretreatment of the cells with homologous IF 20 hr prior to inoculation with SV40 resulted in a dose-dependent decrease in the proportion of T antigen-positive cells at 48 hr postinfection. The kinetics of inhibition by IF of SV40-induced T Ag was studied in both cell types. When IF was added at increasingly later times after SV40 inoculation, there was a progressive reduction of T Ag-positive cells. Penetration and uncoating of SV40 were ruled out as the major site of IF action since the induction of T Ag by infectious DNA extracted from SV40 virions was also sensitive to treatment with IF. In addition, although IF may be added to the cells at a particular time, it is apparent that these cells do not become resistant to viral infection until several hours later. The results suggest that the inhibitory effect of IF is primarily at a step subsequent to the uncoating of SV40.     

Biological properties of simian virus 40 host range mutants lacking the COOH-terminus of large T antigen

Three mutants of simian virus 40 (SV40), with deletions near the 3' end of the A gene, displayed a host range phenotype for growth and virus production in various African green monkey kidney cell lines. The mutants formed plaques in CV-1P cells at 40.5 degrees, in BSC-1 cells at 37 and 40.5 degrees, and in Vero cells at 32, 37, and 40.5 degrees. Virus yields in these three lines were cold sensitive: the burst size was greatest at 40.5 degrees and least at 32 degrees, but some progeny was produced under all conditions examined. Mutant yields never exceeded 10% of wild-type yields under the most permissive conditions (Vero cells at 37 or 40 degrees) and were less than 1% of wild type under the most restrictive conditions (CV-1P cells at 32 degrees). These mutants can be complemented by any SV40 mutant which produces a large T antigen containing a normal COOH-terminus. Mutants whose T antigens could not be transported to the nucleus were most efficient at complementation. Mutant virus production in a line of rhesus monkey kidney cells and in primary cultures of African green and rhesus monkey kidney cells was also substantially below wild type. These mutants were also completely defective for adenovirus helper function. Our data suggest that the host range property and adenovirus helper function represent the same activities of large T antigen.     

Perturbed development of the mouse lens by polyomavirus large T antigen does not lead to tumor formation

To study how the oncogenic process may involve effects on differentiation, we overexpressed an immortalizing oncogene in a developing tissue in transgenic mice. By use of a gene fusion of the alpha A-crystallin promoter to the viral immortalizing oncogene, polyoma large T antigen (PyLT), we created transgenic mice that express PyLT specifically in ocular lens. Expression of large T antigen during embryonic development led to a perturbation in lens development, specifically, an interference with the normal program of fiber cell differentiation. This resulted in microphthalmia, which persisted throughout the life of the animal. Histological analysis revealed impairment of cell elongation, denucleation, and mitotic senescence in both primary and secondary fiber cell differentiation. Strikingly, there was no evidence for hyperplasia or for tumor development in vivo, unlike the consequences of many immortalizing oncogenes on tissues in other transgenic mice. In vitro, however, the developmentally perturbed cells derived from the transgenic lens showed high proliferative capacity. Our results suggest that a primary effect of aberrant expression of an immortalizing gene is an interference with normal tissue development; however, this interference may not necessarily induce proliferation or lead to tumor formation.     

The SV40 A gene product suppresses the adenovirus H5ts125 defect in DNA replication

The adenovirus H5ts125 mutation is a temperature sensitive defect in the structural gene for a DNA-binding protein required for viral DNA replication. Infection of primary African green monkey kidney cells with SV40, followed by superinfection with H5ts125 at the nonpermissive temperature, permits the replication of adenovirus DNA even in the absence of detectable adenovirus DNA binding protein. The SV40 A gene product is required continuously for the replication of H5ts125 adenovirus DNA. The rate of adenovirus DNA replication in SV40 and H5ts125 doubly infected cells at nonpermissive temperature is about one-half that observed for either H5ts125 singly or SV40-H5ts125 doubly infected cells at the permissive temperature. H5ts125 progeny virus are also produced in the SV40-H5ts125-infected cells at the nonpermissive temperature but the yields are only 3% of those observed in doubly infected cells at the permissive temperature. The suppression of the H5ts125 phenotype by SV40 requires a prior infection of primary or secondary African green monkey kidney cells with a high multiplicity of SV40 (50–100 PFU/cell) and H5ts125 DNA replication is optimally observed between 10 and 15 hr after adenovirus superinfection. These observations suggest that the SV40 A gene product could be involved in the initiation of adenovirus DNA replication thereby bypassing the need for the adenovirus DNA-binding protein.