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.     

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.     

Induction of tetraploid DNA content by simian virus 40 is dependent on T-antigen function in the G2 phase of the cell cycle.

Previous experiments with the simian virus 40 mutant tsA357R-K (tsA30) demonstrated a T-antigen function that is required for production of cells with a greater-than-G2-phase DNA content. In this study, temperature shift experiments indicated that the temperature-sensitive function of tsA357R-K, which is necessary for entry into the greater-than-G2 phase, is not required in G1 or S but must be supplied in the G2 phase.     

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 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.     

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.     

Two conditional tsA mutant simian virus 40 T antigens display marked differences in thermal inactivation.

We have characterized the simian virus 40 (SV40) origin-containing DNA (ori-DNA) replication functions of two SV40 conditional mutant T antigens: tsA438 A-V (tsA58) and tsA357 R-K (tsA30). Both tsA mutant T antigens, immunopurified from recombinant baculovirus-infected insect cells, mediated replication of SV40 ori-DNA in vitro to similar extents as did wild-type T antigen in reactions at 33 degrees C. However, at 41 degrees C, the restrictive temperature, while tsA438 T antigen still generated substantial levels of replication products, tsA357 T antigen did not support any detectable DNA synthesis. Furthermore, preincubation for approximately fourfold-longer time periods at 41 degrees C was required to heat inactivate tsA438 T antigen than to heat inactivate tsA357 T antigen. Unexpectedly, results of analyses of the various DNA replication activities of the two mutant T antigens did not correlate with results from ori-DNA replication reactions. In particular, although tsA357 T antigen was incapable of mediating replication at 41 degrees C at all protein concentrations examined, it displayed either wild-type levels or only partial reductions of the several T-antigen replication-associated activities. These data suggest either that tsA357 T antigen is defective in an as yet unidentified replication function of T antigen or that the combination of its partial defects result in a protein that is unable to support replication. The data also show that two conditional mutant T antigens can be markedly different with respect to thermal sensitivity.     

Simian virus 40 A gene function: further characterization and growth of tsA transformed chinese hamster cells

Chinese hamster embryo cells transformed with the tsA 58 mutant of Simian virus 40 express the transformed phenotype at the permissive temperature (33 degrees C or 37 degrees C) and a "normal" phenotype at the nonpermissive temperature (40.5 degrees C). Immunofluorescence and immunoprecipitation of T antigens demonstrated that the "T" antigen (100 K) has an increase rate of synthesis and degradation at 40.5 degrees C. However, the cells continue to replicate at the nonpermissive temperature when assayed by flow cytometry and autoradiography. This DNA synthesis was cellular, not viral, and not owing to an increase in DNA repair. When the cell cycle distributions of G1, S, and G2 + M were assayed by the fraction labeled mitoses method, no differences were evident at the permissive and nonpermissive temperature; however, the doubling time was lengthened at 40.5 degrees C (13 hours vs. 100 hours). These results suggest that at 40.5 degrees C, the tsA transformed cells are cycling and dying. However, if the transformed cells are seeded onto monolayers of normal Chinese hamster cells at 40.5 degrees C, the cells are growth arrested when measured by growth assays, flow cytometry, autoradiography, and immunofluorescence for T antigen. Therefore, growth arrest can be obtained in tsA 58 transformed Chinese hamster cells when cocultured with normal Chinese hamster cells.     

Cold-sensitive growth of simian virus 40 in semipermissive variants of CV1 cells.

Two cell clones were isolated from the simian line CV1, permissive for simian virus 40 (SV40), by selection at low temperature with the tsA239 mutant of SV40. These clones exhibited cold-sensitive semipermissivity to both SV40 virions and SV40 DNA. On the basis of virus yields, their resistance to viral DNA was increased approximately 15 times over that of CV1 cells when the incubation temperature was lowered from 38.5 to 33.5 degrees C. A further 30- to 40-fold resistance increase was exhibited at both temperatures upon infection with SV40 virions. Partial characterization of these clones indicated that the cold sensitivity affected an early function in viral growth, between viral uncoating and the appearance of T-antigen positivity, with a burst-size decrease in all cells at the restricted temperature. This conditional defect appeared to be superimposed upon a temperature-independent uncoating defect, presumably carried in a CV1 subpopulation from which the two clones were ultimately selected.     

The growth of simian virus 40 (SV40) host range/adenovirus helper function mutants in an African green monkey cell line that constitutively expresses the SV40 agnoprotein.

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of the agnoprotein-producing cells.     

Replication of Chinese hamster embryo cells transformed by temperature-sensitive T-antigen mutants of simian virus 40.

Chinese hamster embryo cells transformed by simian virus 40 temperature-sensitive T-antigen mutants replicated when confluent at 40.5 degrees C, regardless of the selection method, selection temperature, or virus strain used.     

SV40-transformed simian cells support the replication of early SV40 mutants

CV-1, an established line of simian cells permissive for lytic growth of SV40, were transformed by an origin-defective mutant of SV40 which codes for wild-type T antigen. Three transformed lines (COS-1, -3, -7) were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replication of pure populations of SV40 mutants with deletions in the early region. One of the lines (COS-1) contains a single integrated copy of the complete early region of SV40 DNA. These cells are possible hosts for the propagation of pure populations of recombinant SV40 viruses.     

Study of liver differentiation in vitro

A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions has been established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of simian virus 40. At the permissive temperature (33 degrees C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost four characteristics of the transformed phenotype at the restrictive temperature (40 degrees C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased, and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized alpha-fetoprotein albumin, and transferrin. At 33 degrees C, the levels of these liver proteins were relatively low. At 40 degrees C the transformed phenotype was lost and the levels of alpha-fetoprotein, albumin, and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of alpha-fetoprotein, albumin, and transferrin was achieved 3-4 d after the upward shift in temperature. The synthesis of alpha-fetoprotein then decreased; the synthesis of albumin and transferrin, however, was maintained. A second phase of albumin and transferrin synthesis was observed in all cultures after 6 d or more at 40 degrees C. Alpha-Fetoprotein, albumin, and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic alpha-fetoprotein, albumin, and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a simian virus 40 tsA255-transformed fetal liver cell line (RLA255-4) reported earlier from this laboratory, responded to glucagon with markedly elevated levels of cyclic AMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the simian virus 40 tsA-transformed fetal liver cells.     

Possible role of the T antigen in inducing chromosome aberrations in SV40 virus-transformed cells

A possible role of the simian virus 40 T antigen in chromosome damages in transformed cells was examined. Two lines of Golden hamster embryonal fibroblasts, transformed by SV40 tsA30 and ts239 mutants (He30 and He239, respectively), were incubated at nonpermissive (40.5-41 degrees C) or permissive (33 degrees C) temperatures. Chromosome aberrations were registered in either subline after 3, 6, 9 and 12 weeks of cultivation under the above conditions. In the both cell lines kept at 33 degrees the frequency of aberrant metaphases and the number of chromosome breaks per cell increased drastically by week 3 of cultivation, and such a state was preserved up to week 12. The frequency of aberrant metaphases in cells cultivated at 41 degrees was maintained at the constant level (He239) or at slightly higher than that in the original culture (He30). The sublines He239, originally incubated at 33 or 40.5 degrees, were then shifted to 40.5 and 33 degrees, respectively. As a result the number of chromosome aberrations either decreased (33----40.5 degrees) or increased (40.5----33 degrees) as early as on day 2, and these patterns were stabilized at the level corresponding to the new conditions. We assayed the induction of DNA breaks in cells, grown at the permissive or nonpermissive temperatures, by using DNA sedimentation in the alkaline sucrose gradient. The DNA sedimentation peaks of cells cultured at 37 and 41 degrees coincided, whereas the DNA of cells cultured at 33 degrees was represented by shorter fragments.     

Abortive transformation of temperature-sensitive mutants of rat 3Y1 cells by simian virus 40: effect of cellular arrest states on entry into S phase and cellular proliferation

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, representing independent complementation groups, cease to proliferate predominantly with a 2n DNA content, at the restrictive temperature (39.8 degrees C) (temperature arrest) or at the permissive temperature (33.8 degrees C) at a confluent cell density (density arrest) (Ohno et al., 1984). We studied the temperature- or the density-arrested cells of these mutants infected with simian virus 40 (SV40) or its mutants affecting large T or small t antigen with respect to kinetics at 39.8 degrees C of entry into S phase and cellular proliferation. Three mutants, 3Y1tsD123, 3Y1tsF121 and 3Y1tsG125, expressed T antigen and entered S phase at 39.8 degrees C from both the arrested states after infection with either wild-type, tsA mutants, or a .54/.59 deletion mutant of SV40, whereas in the density-arrested 3Y1tsH203, expression of T antigen and entry into S phase were inefficient and ts. Following the WT-SV40 induced entry into S phase, the temperature-arrested 3Y1tsD123 detached from the substratum with no detectable increase in cell number, whereas the density-arrested ones completed a round of the cell cycle and then detached. 3Y1tsF121 and 3Y1tsG125 in the both arrested states proliferated through more than one generation. 3Y1tsF121 and 3Y1tsG125 in the density-arrested state infected with tsA mutants once proliferated and then ceased to increase in number as the percentage of T-antigen positive population decreased. These results suggest that wild-type and tsA-mutated large T antigens are able to overcome the cellular ts blocks of entry into S phase in the 3 ts mutants of 3Y1 cells in both the arrested states, and that small t antigen is not required to overcome the blocks. It is also suggested that cellular behaviors subsequent to S phase (viability, mitosis, and proliferation in the following generations) depend on cellular arrest states, on traits of cellular ts defects, and on the duration of large T antigen expression.     

Thermally inactivated simian virus 40 tsA58 mutant T antigen cannot initiate viral DNA replication in vitro.

The mutation in the temperature-sensitive tsA58 mutant T antigen (Ala-438----Val) lies within the presumptive ATP-binding fold. We have constructed a recombinant baculovirus that expresses large quantities of the tsA58 T antigen in infected insect cells. The mutant T antigen mediated simian virus 40 origin-containing DNA (ori-DNA) synthesis in vitro to nearly the same extent as similar quantities of wild-type T antigen at 33 degrees C. However, if wild-type and tsA58 T antigens were heated at 41 degrees C in replication extracts prior to addition of template DNA, the tsA58 T antigen but not the wild type was completely inactivated. The mutant protein displayed greater thermosensitivity for many of the DNA replication activities of T antigen than did the wild-type protein. Some of the replication functions of tsA58 T antigen were differentially affected depending on the presence or absence of ATP during the preheating period. When tsA58 T antigen was preheated in the presence of ATP at 41 degrees C for a time sufficient to completely inactivate its ability to replicate ori-DNA in vitro, it displayed substantial ATPase and normal DNA helicase activities. Conversely, when preheated in the absence of nucleotide, it completely lost both ATPase and helicase activities. Preheating tsA58 T antigen, even in the presence of ATP, led to drastic reductions in its ability to bind to and unwind DNA containing the replication origin. The mutant T antigen also displayed thermosensitivity for binding to and unwinding nonspecific double-stranded DNA in the presence of ATP. Our results suggest that the interactions of T antigen with ATP that are involved in T-antigen DNA binding and DNA helicase activities are different. Moreover, we conclude, consistent with its phenotype in vivo, that the tsA58 T antigen is defective in the initiation but not in the putative elongation functions of T antigen in vitro.