Separation of lytic and transforming functions of the simian virus 40 A region: two mutants which are temperature sensitive for lytic functions have opposite effects on transformation.

Thirty-six of 40 rat cell clones transformed to anchorage independence at low multiplicity of infection by simian virus 40 tsA58 were heat sensitive for continued expression of the transformed phenotype. tsA1499 is an 81-base-pair deletion at 21 map units which is like tsA58 in that it is also heat sensitive for lytic growth, belongs to the A complementation group, and produces rat cell transformants which contain a thermolabile T antigen. Unlike tsA58, however, tsA1499 generated rat cell transformants efficiently at the temperature at which it was lytically defective, and 10 of 17 clones transformed by tsA1499 were cold rather than heat sensitive for the continued maintenance of the transformed phenotype. The lytic and transforming activities of the A region thus appeared to function independently in mutant tsA1499.     

Ability of nonpermissive mouse cells to express a simian virus 40 late function(s)

Mouse cells are fully nonpermissive for simian virus 40 (SV40). Infection does not lead to detectable virus replication. In this report, it was shown, first, that spliced 16S and 19S SV40 late mRNA were present in cytoplasmic and polysomal polyadenylated acid+ RNA preparations from SV40-infected baby mouse kidney cells. The 16S and 19S SV40 late mRNA's produced in infected baby mouse kidney cells were identical to or similar to the 16S and 19S SV40 late mRNA's produced in permissive monkey cells as judged by their S1 mapping patterns performed with the late strand of HpaII-BamHI fragment B and by their sedimentation patterns in a sucrose gradient. It was also shown that the 16S late mRNA from infected baby mouse kidney cells could be translated into a polypeptide which was identical to or similar to virion protein VP1 in every aspect examined, including the patter of peptide mapping by limited proteolysis. Second, we reported that mouse kidney cells produced detectable, although low, levels of SV40 virion protein VP1, as shown by the sodium dodecyl sulfate-polyacrylamide gel autoradiogram of [35S]methionine-labeled proteins immunoprecipitated by a rabbit antiserum directed against SV40 virion proteins. Third, it was reported that infected baby mouse kidney cells produced late mRNA's either (i) when the infection was done at a restrictive temperature with the nonleaky tsA58 mutant or (ii) in cells treated with 100 microgram of cycloheximide per ml, in which large T antigen synthesis was inhibited by more than 99.9%. This suggested that large T antigen was not required for the synthesis of late mRNA in mouse cells.     

Evolutionary variants of simian virus 40: Cellular DNA sequences and sequences at recombinant joints of substituted variants.

A correlation is presented of all the presently known asymmetries of the purple membrane from Halobacterium halobium with its sidedness in the cell.     

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.     

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.     

Quantitative correlation between simian virus 40 T-antigen synthesis and late viral gene expression in permissive and nonpermissive cells

The time-course of intranuclear Simian virus 40 (SV40) tumor (T) antigen synthesis and accumulation in permissive CV1 monkey cells and nonpermissive 3T3 mouse cells has been studied by immunofluorescence and cytofluorometry. CV1 cells accumulate T antigen continuously over a period of 48 h after infection, whereas in 3T3 cells the T-antigen content remains about constant and at a comparatively low level. Only those CV1 cells which have attained a threshold concentration of intranuclear T antigen synthesize viral capsid proteins (V antigen). In nonpermissive 3T3 cells, the T-antigen threshold value for the onset of V-antigen synthesis is higher than in CV1 cells and is never reached by infected cells. However, 3T3 cells microinjected with sufficient amounts of SV40 DNA easily surpass this value and behave permissively.     

Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.

The thermolabile large T antigen, encoded by the simian virus 40 early-region mutant tsA58, was used to establish clonal cell lines derived from rat embryo fibroblasts. These cell lines grew continuously at the permissive temperature but upon shift-up to the nonpermissive temperature showed rapidly arrested growth. The growth arrest occurred in either the G1 or G2 phase of the cell cycle. After growth arrest, the cells remained metabolically active as assayed by general protein synthesis and the ability to exclude trypan blue. The inability of these cell lines to divide at the nonpermissive temperature was not readily complemented by the exogenous introduction of other nuclear oncogenes. This finding suggests that either these genes establish cells via different pathways or that immortalization by one oncogene results in a finely balanced cellular state which cannot be adequately complemented by another establishment gene.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen

Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 degrees C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 degrees C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21(waf1), milk fat globule membrane protein E8, heat-responsive protein 12, and selenoprotein P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen.     

Comparison of two viable variants of simian virus 40.

The DNAs of two viable strains of simian virus 40, 776 and 777, have been compared by using restriction endonucleases. Differences between the two strains were detected at five separate points on the simian virus 40 genome. One of these differences, in the region of DNA coding for the major viral coat protein, was confirmed by tryptic peptide analysis of coat proteins from the two strains. Some physiological differences between the two strains were examined and can, in general, be explained by differences observed between the DNAs of the two strains. In addition, defective variants derived from strain 777 interfere more efficiently with the replication of strain 777 than with the replication of strain 776.     

Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle.

We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.     

Functional implications of oligomerization of simian virus 40 large T antigen during lytic virus infection.

The formation of oligomers of simian virus 40 (SV40) large T antigen in SV40-infected and -transformed monkey cells was analyzed by sucrose density gradient centrifugation. The overall distribution of total T antigen during lytic infection showed mainly low-molecular-weight forms (monomers and dimers) in the early phase (10 h postinfection) and an increase in the number of oligomers in the late phase of the lytic cycle (36 h postinfection), indicating an accumulation of these final products. In contrast, studying the conversion of newly synthesized T antigen into oligomers by appropriate pulse-chase radiolabeling of infected cells revealed that this processing decelerates considerably during the late phase of infection. This mechanism can be reaccelerated by blocking DNA replication with aphidicolin. Since none of these results could be obtained by using synchronized SV40-transformed monkey cells (COS-1), these observations are compatible with the idea that the process of T antigen oligomerization may be involved in viral, but not in cellular, DNA synthesis.     

Failure of simian virus 40 small t antigen to disorganize actin cables in nonpermissive cell lines.

Mouse C3H 10T1/2 cell lines expressing the simian virus 40 (SV40) small t antigen were obtained by cotransfection of pSV2neo and plasmids which encode small t. Cell lines derived from two plasmids which encode small t in the absence of stable deletion fragments of the large T antigen were morphologically normal and grew to slightly higher saturation densities in low serum than control cell lines. Unexpectedly, the clones had highly organized actin cables, as did parental 10T1/2 cells infected with wild-type SV40. These observations and comparisons of rat F111 cells infected with either polyomavirus or SV40 suggest that the SV40 small t antigen does not directly affect cytoskeletal organization.     

Emergence of simian virus 40 variants during serial passage of plaque isolates

Three serial passage series of simian virus 40 (SV40) in CV-1 cells were initiated by infection directly from the same wild-type plaque isolate, three series were initiated by infection with another plaque isolate, and two series were initiated with each of two other plaque isolates. Aberrant SV40 genomes were not detected in any of the passage series until after the fifty undiluted passage, and each series generated a different array of variant genomes. The results show that the variants were not present in the original plaque isolates but, instead, were randomly generated during subsequent high-input multiplicity passages. Although many of the aberrant viral genomes in each passage series contained reiterations of the SV40 origin of replication and some also contained host cell sequences, there was no indication that SV40 is predisposed toward generating any particular variant.     

Recurring defective variants of simian virus 40 containing monkey DNA segments.

Four independently and newly isolated defective variants of simian virus 40 have been characterized. All four are very similar, if not identical, to two previously and independently isolated variants (Wakamiya et al., J. Biol. Chem. 254:3584-3591, 1979; J. Papamatheakis, E. Kuff, E. Winocour, and M. F. Singer, J. Biol. Chem. 255:8919-8927, 1980). The documented similarities include restriction endonuclease maps and the presence of the same monkey DNA segments covalently linked to simian virus 40 DNA sequences. Each of the newly described variants was first detected upon serial passaging of wild-type simian virus 40 at a high multiplicity of infection at 33 degrees C as recently described (M. F. Singer and R. E. Thayer, J. Virol. 35:141-149, 1980). A variety of experiments support the idea that the various isolates were independent and do not reflect inadvertent cross-contamination. Two of the new isolates arose during passage of wild-type strain 777 virus in BSC-1 cells, one during passage of strain 776 in BSC-1 cells, and one during passage of strain 776 in primary African green monkey kidney cells. The two variants obtained after passage of strain 776 were shown to contain a particular recognition site for restriction endonuclease MboII within their simian virus 40 DNA segments, as do the two previous isolates. This site is not present in wild-type strain 776 DNA but is shown here to be present in wild-type strain 777 DNA. The surprising recurrence of closely related variants and particularly the unexpected presence of the endo R.MboII site in variants derived from passaging strain 776 suggest that these variants may arise by mechanisms other than recombination between the initial infecting viral genome and the host DNA.     

Accumulation of cells with 4N DNA content at nonpermissive temperature in rat embryo diploid cells transformed by tsA mutant of simian virus 40

Primary rat embryo cells were transformed by a tsA mutant (tsA640) of simian virus 40 (SV40). Proliferation of all four independent diploid transformants was suppressed at a nonpermissive temperature (40.3 degrees C), being accompanied by a marked increase in the fraction of cells with a 4N DNA content (a 4N peak in the flow cytofluorogram). However, in this case, the fraction of cells with a 2N DNA content (a 2N peak in the flow cytofluorogram) was preserved. Both effects (suppression of proliferation and increase in the 4N peak) diminished when transformed cells were superinfected with wild-type SV40. The increased 4N peak was preserved, albeit not completely, for at least 24 hours, when cells were further incubated in the presence of hydroxyurea at the nonpermissive temperature. On the other hand, the preserved 2N peak all but disappeared within 24 hours, when cells were further incubated in the presence of colcemid at the nonpermissive temperature. These results suggest that the thermolabile large T antigen of SV40 directly or indirectly induces an accumulation of cells with a 4N DNA content, at the nonpermissive temperature, by prolonging the G2 (and/or late S) period.     

Cellular proteins associated with simian virus 40 early gene products in newly infected cells.

Immunoprecipitates of extracts of simian virus 40-infected permissive monkey kidney cells contained two proteins with molecular weights of 56,000 and 32,000 (52K and 32K) in addition to the known viral early gene products. Immunoprecipitates of cells infected with the 0.54-0.59 deletion mutants that lack the viral 17K gene product did not contain thhe 56K and 32K proteins. The additional proteins appeared in immunoprecipitates of deletion mutant extracts if unlabeled extracts of wild-type-infected cells were added before addition of antiserum. The proteins can also be identified in uninfected cells by co-precipitation with unlabeled viral proteins. Thus, it appears that the 56K and 32K proteins are cellular products that associate with the viral proteins, the 17K in particular, and are indirectly immunoprecipitated by anti-tumor serum.     

Replication of ultraviolet-irradiated simian virus 40 in monkey kidney cells

This paper extends the concepts of linkage and control, previously studied in single phase allosteric and polysteric systems, to multiple phase (polyphasic) systems. In particular, a study has been made of the dependence of the solubility of sickle cell hemoglobin on oxygen partial pressure. Phase diagrams are obtained from observations of birefringence changes of hemoglobin solutions in a thin film optical cell. The effects of temperature and pH are found to be correlated largely with oxygen binding curves for non-gelling solutions. This suggests only small enthalpy and proton release changes for the gelation process. Variable time delays for the onset of birefringence were observed for partial deoxygenation of a fully oxygenated sample. The reciprocal of the time delay depends on a high power of the supersaturation ratio. The nucleation kinetics are, thereby, similar to those found in fully deoxygenated solutions in temperature-jump studies. Oxygen binding curves for non-gelling solutions of sickle cell hemoglobin were used in conjunction with the phase diagram results to evaluate oxygen binding curves for the polymer gel. Account was taken of the water content of the gel and of the large non-ideality of the solution. Analysis of the phase diagram data based on polyphasic linkage relationships suggests that some reversible oxygen-binding by the gel is present. The difference in oxygen binding between solution and gel obtained in this way is similar to that found by Hofrichter (1979) for carbon monoxide.