Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells.

The kinetics of host cellular DNA stimulation by simian virus 40 (SV40) tsA58 infection was studied by flow microfluorometry and autoradiography in two types of productively infected monkey kidney cells (AGMK, secondary passage, and the TC-7 cell line). Prior to infection, the cell populations were maintained predominantly in G0-G1 hase of the cell cycle by low (0.25%) serum concentration. Infection of TC-7 or AGMK cells by wild-type SV40, viable deletion mutant dl890, or by SV40 tsA58 at 33 degrees C induced cells through S phase after which they were blocked with a 4N DNA content in the G2 phase. The infection of TC-7 cells by tsA58 at 41 degrees C, which was a nonpermissive temperature for viral DNA replication, induced a round of cell DNA synthesis in approximately 30% of the cell population. These cells proceeded through S phase but then re-entered the G1 resting state. In contrast, infection of AGMK cells by tsA58 at 41 degrees C induced DNA synthesis in approximately 50% of the cells, but this population remained blocked in the G2 phase. These results indicate that the mitogenic effect of the A gene product upon cellular DNA is more heat resistant than its regulating activity on viral DNA synthesis and that the extent of induction of cell DNA synthesis by the A gene product may be influenced by the host cell.     

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.     

Ribosomal RNA synthesis in spermatogonia and Sertoli cells of the mouse testis

Simultaneously cloned monkey CV-1 cell sublines were found to differ in morphology, cloning efficiency, chromosome number, and sensitivity to SV40 virion productive infection. A fibroblast-like clone, FC7, when compared with an epithelioid clone, TC7, had a lower mean chromosome number and was resistant to SV40 virion infection. Virion adsorption and penetration were similar in both the FC7 and TC7 cells, and both cell types were equally sensitive to first cycle SV40 DNA infection. As the subdiploid mean chromosome number of the FC7 cells increased with passage time toward the TC7 subtetraploid number, the FC7 cells became more sensitive to virion infection. This host gene-dosage effect on SV40 productive infection suggests that a monkey cell function participates in the SV40 uncoating and/or viral genome activation process.     

SV40-immortalized human fibroblasts as a source of SV40 infectious virions

Human fibroblasts immortalized by Simian Virus 40 (SV40) are widely employed for cell and molecular biology model of study. Indeed, SV40 transmission to humans was believed to occur only under exceptional situations. The oncogenic potential of SV40 in laboratory animals is well established, whereas its involvement in human carcinogenesis is still a matter of active investigations. A recent report links SV40 exposure with the development of a brain tumor in a laboratory researcher. In previous studies, episomal viral DNA was detected in SV40 stably transformed and immortalized fibroblast cell lines. In this study, we report molecular and biological characterizations of SV40 DNA in human fibroblast cells. Our results indicate that SV40 is able to establish a persistent infection in long-term immortalized human fibroblasts, resulting in the production of an infectious viral progeny, which is able to infect both monkey and human cells. These data indicate that SV40-immortalized human fibroblasts may represent a source of SV40 infection. To avoid the SV40 infection, careful attention should be given by operators to this SV40-cell model of study.     

Mutations near the carboxyl terminus of the simian virus 40 large tumor antigen alter viral host range.

We report the characterization of three mutants of simian virus 40 with mutations that delete sequences near the 3' end of the gene encoding large tumor antigen (T antigen). Two of these mutants, dl1066 and dl1140, exhibit an altered viral host range. Wild-type simian virus 40 is capable of undergoing a complete productive infection on several types of established African green monkey kidney lines, including BSC40 and CV1P. dl1066 and dl1140 grow on BSC40 cells at 37 degrees C. However, both mutants fail to form plaques on BSC40 cells at 32 degrees C or on CV1P cells at any temperature. These mutants are capable of replicating viral DNA in the nonpermissive cell type, indicating a defect in an activity of T antigen not related to its replication function. Furthermore this defect can be complemented in trans by the wild type or by a variety of DNA replication-negative T antigen mutants, so long as they produce a normal carboxyl-terminal region of the molecule. Our data are consistent with the hypothesis that the C-terminal region of T antigen constitutes a functional domain. We propose that this domain encodes an activity that is required for simian virus 40 productive infection on the CV1P cell line, but not on BSC40.     

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization.     

Regulation of SV40-induced cell division and tumor antigen by dibutyryl adenosine 3′-5′-monophosphate

Simian virus 40 (SV40) induces cell division in microcultures of sparsely plated nongrowing mouse BALB/3T3 cells during acute infection at moderate multiplicities of infection (MOI = 10–100). The infected cells are killed when a MOI of 1,000 is used. SV40 tumor (T) antigen is synthesized in the infected cells, but viral DNA, virion antigen, and progeny virions are not synthesized (abortive infection). The addition of exogenous dibutyryl adenosine 3′-5′-monophosphate (dbcAMP) at the time of infection stimulates the SV40-induced cell division at all MOI and inhibits SV40-induced cell death at high MOI. The percentage of T antigen-positive cells, as monitored by immunofluorescence, is also increased by the addition of dbcAMP at the time of infection. This regulation of SV40-induced cell division and T antigen formation by exogenous dbcAMP occurs within the first 6 hr after infection at 37° C and is dependent upon both the MOI and the concentration of added dbcAMP. The addition of dbcAMP to productively infected TC7 monkey cells has little effect on the SV40-induced cell death or T antigen formation.     

SV40 vector with early gene replacement efficient in transducing exogenous DNA into mammalian cells.

An early replacement SV40 vector, SV40-Mu beta, was constructed by replacing part of the early genes of the virus with mouse interferon-beta (IFN-beta) cDNA. Upon transfection of COS-7 cells with this DNA, transducing viral particles were produced, which could infect various cells and cause efficient production of mouse IFN-beta. The viral stock contained no detectable wild-type SV40. The IFN production after the virus infection was very high in monkey kidney cells, but less so in human epithelial cells, and low in mouse, pig, hamster cells and in human lymphocytes. The efficiency of introduction of the DNA to monkey kidney cells was compared with that by the calcium phosphate precipitation method, and the viral vector was found to be more efficient by a factor of several tens to hundreds.     

Survival and mutagenesis of ultraviolet irradiated simian virus 40 in foetal human fibroblasts

Survival and mutagenesis of UV-irradiated, temperature-sensitive simian virus 40 mutants (SV40) have been studied after infection of human fibroblasts. Survival of the viral progeny obtained after 6,8 or 10 days at permissive temperature decrease as a function of the UV-dose delivered to the virus. In cels which have been pretreated with 10 Jm-2 of UV 24 hours before infection, progeny survival was increased as compared to survival in control cells. The reactivation factor varies from one to ten, depending on the number of lytic cycles carried out at permissive temperature. The level of mutation frequency, as measured by the reversion from a temperature sensitive growth phenotype towards a wild type phenotype, increases with the dose of UV-irradiation given to the virus. Moreover, the mutation frequency is increased in the viral progeny produced in UV-irradiated human cells. Similar experiments carried out with SV40-transformed human fibroblasts, which constitutively express SV40 T antigen, gave comparable results. These experiments show that, as in monkey cells, a new error-prone recovery pathway can be induced by pretreating human cells with UV-light before infection.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

Effect of a tsA mutation of simian virus 40 late gene expression: variations between host cell lines

Infection of AGMK or CV-1 cells by the early simian virus 40 mutant tsA58 at the permissive temperature (32 degrees C) followed by a shift to the nonpermissive temperature (41 degrees C) caused a substantial decrease in the levels of late viral RNA in the cytoplasm of AGMK cells but not CV-1 cells. At the translational level, this depression of late viral RNA levels was reflected by a decrease in late viral protein synthesis. Thus, in AGMK cells, an early region gene product (presumably large T-antigen) appeared to be continuously required for efficient expression of the late viral genes. In contrast, late simian virus 40 gene expression, once it is initiated in CV-1 cells, continued efficiently regardless of the tsA mutation. The difference in expression of the late simian virus 40 genes in these tsA mutant-infected monkey kidney cell lines may reflect a difference in host cell proteins which regulate viral gene expression in conjunction with early viral proteins.     

Simian virus 40 induces multiple S phases with the majority of viral DNA replication in the G2 and second S phase in CV-1 cells

The infection of permissive monkey kidney cells (CV-1) with simian virus 40 induces G1 growth-arrested cells into the cell cycle. After completion of the first S phase and movement into G2, mitosis was blocked and the cells entered another DNA synthesis cycle (second S phase). Growth-arrested CV-1 cells replicated significant amounts of viral DNA in the G2 phase with the majority of synthesis occurring during the second S phase. When mimosine-blocked (G1/S) infected cells were released into the cell cycle, a major portion of the viral DNA was detected in G2 with the largest accumulation in the second S phase. The total DNA produced per infected cell was 10-12C with approximately 0.5-2C of viral DNA replicated per cell. Therefore the majority of the DNA per cell was cellular, 4C from the first S phase and approximately 4-6C from the second cellular synthesis phase.     

Flow cytometry analysis of early DNA content changes in human and monkey cells following infection with simian virus 40

Simian virus 40 (SV₄₀) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfects (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (～60%) of the cells were blocked in the G₂ + M phase of the cell cycle. At later time intervals, an increase in the G₁ population was demonstrated. The two monkey cell lines responded to SV₄₀ virus with an accumulation of cells in the G₂ + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV₄₀ virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transformin) cells infected with SV₄₀ virus.     

Preferential replication of a class of host-substituted defective simian virus 40 variants at low temperature

The host-substituted variant termed CVP8/1/P2 (EcoRI res) was first isolated several years ago after serial passage of simian virus 40 strain 777 on BSC-1 cells at 37 degrees C. When BSC-1 are coinfected with wild-type simian virus 40 strain 777 and variant CVP8/1/P2 (EcoRI res), the variant rapidly becomes the dominant species produced, often representing as much as 80% of the total DNA I synthesized after infection. We present evidence that the replicative advantage of the variant was increased when the infection was carried out at 33 rather than 37 degrees C. Also described are nine new and independent serial passage experiments carried out at 33 degrees C with several purified wild-type virus stocks, including strain 776, and both BSC-1 and primary African green monkey kidney cells. In each series variants related to CVPs/1/P2 (EcoRI res) were detected in the progeny viral genomes after four serial passages. Hybridization data suggest that at least some of these variant DNA I molecules contain simian virus 40 DNA sequences, monkey alpha-component DNA sequences (highly repetitive), and the infrequently reiterated monkey DNA sequences found in CVP8/1/P2 (EcoRI res), all covalently linked as in CPV8/1/P2 (EcoRI res). It appears that this type of variant emerges with some frequency during infection and is then preferentially replicated at 33 degrees C, thereby becoming readily detectable in passaged stocks. A variety of control experiments indicated that the repeated emergence of similar, if not identical, variants is unlikely to be the result of inadvertent cross-contamination or the presence of detectable amounts of the variant in the plaque-purified viral stocks.     

Effect of UV-irradiation on DNA replication of the parvovirus minute-virus-of-mice in mouse fibroblasts.

The effect of UV-irradiation on the conversion of the single-stranded DNA of the parvovirus Minute-Virus-of-Mice (MVM) to duplex Replicative Forms (RF) was studied after infection of mouse A9 fibroblasts. UV-irradiation of the virus prior to infection of unirradiated cells resulted in a dose-dependent, single-hit, inhibition of RF formation. Restriction fragment analysis indicated that this inhibition could be ascribed to the introduction of absolute blocks which prevent elongation of the newly synthesized complementary strand. Cell exposure to UV-light prior to infection with UV-irradiated MVM enhanced the fraction of input viral DNA which was converted to RF. This enhancement required de novo protein synthesis during the interval between cell irradiation and virus infection. These results suggest that DNA replication constitutes a target in the viral life cycle that leads to the UV-enhanced Reactivation of virus survival, however, they do not permit us to identify the step of RF formation which is enhanced in UV-pretreated cells.     

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.     

Transformation of human fibroblasts by ionizing radiation, a chemical carcinogen, or simian virus 40 correlates with an increase in susceptibility to the autonomous parvoviruses H-1 virus and minute virus of mice.

Morphologically altered and established human fibroblasts, obtained either by 60Co gamma irradiation, treatment with the carcinogen 4-nitroquinoline 1-oxide, or simian virus 40 (SV40) infection, were compared with their normal finite-life parental strains for susceptibility to the autonomous parvoviruses H-1 virus and the prototype strain of minute virus of mice (MVMp). All transformed cells suffered greater virus-induced killing than their untransformed progenitors. The cytotoxic effect of H-1 virus was more severe than that of MVMp. Moreover, the level of viral DNA replication was much (10- to 85-fold) enhanced in the transformants compared with their untransformed parent cells. Thus, in this system, cell transformation appears to correlate with an increase in both DNA amplification and cytotoxicity of the parvoviruses. However, the accumulation of parvovirus DNA in the transformants was not always accompanied by the production of infectious virus. Like in vitro-transformed fibroblasts, a fibrosarcoma-derived cell line was sensitive to the killing effect of both H-1 virus and MVMp and amplified viral DNA to high extents. The results indicate that oncogenic transformation can be included among cellular states which modulate permissiveness to parvoviruses under defined growth conditions.