Simian Virus 40-Host Cell Interactions. I. Temperature-Sensitive Regulation of SV40 T Antigen in 3T3 Mouse Cells Transformed by the ts*101 Temperature-Sensitive Early Mutant of SV40

BALB/3T3 and Swiss/3T3 mouse cells transformed at permissive temperature (33 C) by the early temperature-sensitive mutant of simian virus 40 (SV40), ts(*)101, exhibited a temperature-dependent modulation of SV40 tumor (T) antigen as assayed by immunofluorescence. The percentage of T antigen-positive nuclei in ts(*)101 transformed cells was reduced at restrictive temperature (39 C) when compared to 33 C and to wild-type SV40 transformed cells at either 33 C or 39 C. The percentage of T antigen-positive nuclei in ts(*)101 transformed cells returned to the 33 C control level when the cells were shifted from 39 to 33 C. The ts(*)101 transformed cells could be superinfected with wild-type, but not ts(*)101, virions at 39 C as assayed by an increase in T antigen-positive nuclei.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. I. Rescue of a Simian Virus 40 Temperature-Sensitive Mutant by Passage in Permissive Transformed Monkey Lines

Passage of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 at the permissive temperature in each of three permissive lines of SV40-transformed monkey CV1 cells resulted in the emergence of temperature-insensitive virus, which plated like wild-type SV40 at the restrictive temperature on normal CV1 cells. In independent experiments, the amount of temperature-insensitive virus that appeared after passage on transformed cells was from 10(3)- to 10(6)-fold greater than the amount of ts-revertant virus that appeared after an equal number of passages in nontransformed CV1 cells. The virus rescued by passage on transformed cells bred true upon sequential plaque purification, plated on normal CV1 cells with single-hit kinetics at the restrictive temperature, and displayed no selective growth advantage on transformed cells compared to non-transformed cells. Hence, the reversion of the ts phenotype is neither due to complementation effects nor to the selection of preexisting revertants, which grow better on transformed cells. In the accompanying article (T. Vogel et al., J. Virol. 24:541-550, 1977), we present biochemical evidence that the rescue of tsD202 mediated by passage on transformed cells is due to recombination with the resident SV40 genome. Parallel experiments in which tsA, tsB, and tsC SV40 mutants were passaged in each of the three permissive lines of SV40-transformed monkey cells resulted in either only borderline levels of rescue (tsA mutants) or no detectable rescue (tsB and tsC mutants). Evidence is presented that the resident SV40 genome of the transformed monkey lines is itself a late ts mutant, and we suggest that this accounts for the lack of detectable rescue of the tsB and tsC mutants. We furthermore suggest that the borderline level of rescue observed with two tsA mutants is related to a previous finding (Y. Gluzman et al., J. Virol. 22:256-266, 1977) which indicated that the resident SV40 genome of the permissive transformed monkey cells is defective in the function required for initiation of viral DNA synthesis.     

Integration of SV40 DNA into the cell genome and viral mutagenesis

Integration of DNA of a temperature-sensitive SV40 mutant (tsA239) into the cell genome was studied. The viral A gene (the oncogene) encodes the tumour T antigen which is ts in the mutant and is devoid of mutagenic and transforming activity under non-permissive conditions (40 degrees C). Clones of Chinese hamster cells infected by tsA239 mutant were analysed. Those infected by wild-type SV40 served as controls. As shown by dot-hybridization, SV40 DNA was detected in cells of 14 out of 18 clones infected by tsA mutant and incubated at 40.5 degrees C, and in all 20 clones infected by tsA mutant and incubated under permissive conditions (33 degrees C), the difference between the two groups being insignificant (p greater than 0.05). By means of blot-hybridization it was established that viral DNA was integrated into the cell genome of all 12 clones analysed, belonging to the three experimental series: infection by tsA mutant, incubation at 40.5 and 33 degrees C, infection by wt SV40, incubation at 40.5 degrees C. The number of integration sites ranged from one to four in different clones. Integration of SV40 DNA in tandems was observed. The data presented allow to conclude that integration per se does not play a crucial role in determining the mutagenic and transforming effect of the virus. Obviously, what matters is the activity of viral oncogene product - the T antigen.     

Integration of the simian virus 40 genome into cellular DNA in temperature-sensitive (N) and temperature-insensitive (A) transformants of 3T3 rat and Chinese hamster lung cells

We studied the pattern of integration of the simian virus 40 (SV40) genome into the cellular DNA of N-transformants (temperature sensitive) and A-transformants (temperature insensitive) derived from 3T3-Fisher rat and Chinese hamster lung cells. The SV40 DNA was covalently linked to the cellular DNA in both types of transformants. In the rat cells, most N-transformants contained SV40 sequences integrated at a single site; most A-transformants contained SV40 sequences integrated at two to five sites. In the Chinese hamster cells, no significant correlation between the number of integration sites and the phenotype of the transformant was found; one of three integration sites were observed for both the N- and A-transformants. Single copies and tandem repeats of SV40 sequences were observed in A- and N-transformants derived from rat cells. A-transformants arise neither by amplification of the SV40 genome nor by integration at a unique site.     

Isolation and characterization of a heat-inducible simian virus 40 mutant.

We have isolated a new type of temperature-sensitive mutant of simian virus 40 (SV40) that is capable of productive infection in permissive cells but not of maintenance of viral DNA integration in transformed cells at the conditional temperature. Virus development is induced when cells transformed by this mutant are shifted to temperatures above 39 degrees C, but is not induced below this temperature. The plaque-purified, temperature-sensitive mutant virus confers heat inducibility to new host cells, indicating that the conditional function is a property of the viral genome. Unlike previously described temperature-sensitive SV40 mutants, in (ts)-1501 is capable of productive infection in permissive cells at the conditional temperature. The morphology, growth, and oncogenicity of in (ts)-1501-transformed cells at 37 degrees C are similar to those of cell lines transformed by wild-type SV40. HK10-c2(in(ts)-1501), a cloned cell line, transformed at 37 degrees C by the mutant virus, exhibits a transient increase in DNA synthesis before cell death at the conditional temperature. Many properties of in(ts)-1501 are analogous to those of the heat-inducible mutants of bacteriophages in which a heat-inactivated protein is responsible for the stable integration of the prophage in the bacterial chromosome.     

Transformation of primary rat kidney cells by fragments of simian virus 40 DNA.

Linear simian virus 40 (SV40) DNA molecules of genome length and DNA fragments smaller than genome length when prepared with restriction endonucleases and tested for transforming activity on primary cultures of baby rat kidney cells. The linear molecules of genome length (prepared with endonucleases R-EcoRI, R-BamHI, and R-HpaII or R-HapII), a 74% fragment (EcoRI/HpaII or HapII-A), and a 59% fragment (BamHI/HapII-A) could all transform rat kidney cells with the same efficiency as circular SV40 DNA. All transformed lines tested contained the SV40-specific T-antigen in 90 to 100% of the cells, which was taken as evidence that the transformation was SV40 specific. The DNA fragments with transforming activity contained the entire early region of SV40 DNA. Endo R-HpaI, which introduced one break in the early region, apparently inactivated the transforming capacity of SV40 DNA, since no transformation was observed with any of the three HpaI fragments tested. Attempts were made to rescue infectious virus from some of the transformed lines by fusion with permissive BSC-1 cells. Infectious virus was only recovered from the cells transformed by circular form I DNA. No infectious virus could be isolated from any of the other types of transformed cells.     

Characteristics of the Syrian Hamster Ribonucleic Acid Present in Cells Transformed by Polyoma, Simian Virus 40, or Adenovirus 12

Ribonucleic acids of known specific activities were separately prepared from hamster cells transformed by polyoma, simian virus 40 (SV40), and adenovirus 12. When increasing amounts of these labeled ribonucleic acids were added to nitrocellulose filters containing immobilized (14)C-hamster deoxyribonucleic acid (DNA), about 5% of the DNA sites were saturated in each case. An additive saturation-hybridization experiment indicated that a portion of the 5% of the hamster DNA transcribed in SV40-transformed cells was also transcribed in polyoma-transformed cells. In addition, a separate region of the hamster genome was also transcribed in polyoma-transformed cells.     

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.     

Properties of permissive monkey cells transformed by UV-irradiated simian virus 40.

African green monkey cells (CV1 line) were infected with UV-irradiated simian virus 40 (SV40), and permissive lines of stably transformed cells were established. These cell lines display the SV40 T-antigen and the growth characteristics typical of nonpermissive transformed cells (e.g., reduced cell density inhibition, reduced serum dependence, ability to overgrow normal cells, and colony formation in soft agar). The level of permissiveness to superinfecting SV40 is fully comparable with that of nontransformed CV1 and BSC-1 lines. The transformed monkey lines also support SV40 plaque production under agar. By Cot analysis, the transformed permissive cells contain, on an average, 1 to 2 SV40 genome equivalents, and the majority of the viral sequences are associated with the high-molecular-weight cellular DNA. No spontaneous production of infectious SV40 has been observed. The transformed permissive monkey cells failed to support the replication of SV40 tsA mutants at the restrictive temperature. To account for this, it is suggested that the gene A product has separate functions for transformation and initiation of viral DNA synthesis, and only the former function is expressed in the transformed permissive monkey cells.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.     

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

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

Acquisition of Sequences Homologous to Host Deoxyribonucleic Acid by Closed Circular Simian Virus 40 Deoxyribonucleic Acid

The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA.     

Isolation and characterization of defective simian virus 40 genomes which complement for infectivity.

A new variant of simian virus 40 (EL SV40), containing the complete viral DNA separated into two molecules, was isolated. One DNA species contains nearly all of the early (E) SV40 sequences, and the other DNA contains nearly all of the late (L) viral sequences. Each genome was encircled by reiterated viral origins and termini and migrated in agarose gels as covalently closed supercoiled circles. EL SV40 or its progenitor appears to have been generated in human A172 glioblastoma cells, as defective interfering genomes during acute lytic infections, but was selected during the establishment of persistently infected (PI) green monkey cells (TC-7). PI TC-7/SV40 cells contained EL SV40 as the predominant SV40 species. EL SV40 propagated efficiently and rapidly in BSC-1, another line of green monkey cells, where it also formed plaques. EL SV40 stocks generated in BSC-1 cells were shown to be free of wild-type SV40 by a number of criteria. E and L SV40 genomes were also cloned in the bacterial plasmid pBR322. When transfected into BSC-1 cell monolayers, only the combination of E and L genomes produced a lytic infection, followed by the synthesis of EL SV40. However, transfection with E SV40 DNA alone did produce T-antigen, although at reduced frequency.     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.     

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.     

Spontaneous Virus Production by Clonal Lines of Simian Virus 40-Transformed Cells and Effects of Superinfection by Deoxyribonucleic Acid from Mutant Simian Virus 40 Strains

Small amounts of infectious simian virus 40 (SV40) were recovered from parental cultures of SV40-transformed human embryonic lung (WI38 Va13A) cells, from 12 primary clones, from 17 secondary clones, and from 18 tertiary clones. The cloning experiments demonstrated that the capacity for spontaneous virus production is a hereditary property of WI38 Va13A cells. Infectious virus was not recovered from every clone at every passage. Repeated trials at different passage levels were necessary to detect virus production. Approximately one in 10(5) to 10(6) of the cells of the clonal lines initiated plaque formation when plated on the CV-1 line of African green monkey kidney cells. No increase in infectious center formation was observed after the clonal lines were treated with bromodeoxyuridine, iododeoxyuridine, or mitomycin C or after heterokaryon formation of treated cells with CV-1 cells. The clonal lines of WI38 Va13A cells were susceptible to superinfection by SV40 deoxyribonucleic acid (DNA). To determine whether only those cells which spontaneously produced virus supported the replication of superinfecting SV40 DNA, cultures were infected with DNA from a plaque morphology mutant and a temperature-sensitive mutant of SV40. After infection by SV40 DNA, approximately 100 to 4,400 times more transformed cells formed infectious centers than were spontaneously producing virus. To determine whether the resident SV40 genome or the superinfecting SV40 genome was replicating, infectious centers produced by SV40 DNA-infected WI38 Va13A cells on CV-1 monolayers were picked and the progeny virus was analyzed. Only the superinfecting SV40 was recovered from the infectious centers, indicating that in the majority of superinfected cells the resident SV40 was not induced to replicate.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. II. Biochemical Evidence for Genetic Exchange

The genome of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 rescued by passage on transformed permissive monkey lines (see accompanying paper [Y. Gluzman et al., J. Virol. 24:534-540, 1977]) was analyzed by restriction endonuclease cleavage mapping to obtain biochemical evidence that the rescue of the ts phenotype results from recombination with the resident SV40 genome of the transformed cell. It was demonstrated that the endonuclease R. HaeIII cleavage site, which is located at 0.9 map unit in the standard viral genome (and which is in the proximity of the known map position of the tsD lesion), is missing in the DNAs of the parental tsD202 virus and of three independent revertants of tsD202. In contrast, this cleavage site was shown to be present in the DNAs of four out of five independently derived rescued D202 populations and in the DNA of the SV40 strain, 777, used to transform the monkey cells. Comparison of the endonuclease R. Hin(II + III) cleavage patterns of SV40 strain 777 DNA and tsD202 DNA revealed differences in the electrophoretic mobilities of Hin fragments A, B, and F. However, the corresponding Hin fragments from all four rescued D202 genomes were identical in their mobilities to those of tsD202 DNA, indicating that these regions of the rescued D202 genome are characteristic of the tsD202 parent. We conclude, therefore, that the genome of the rescued D202 virus is a true recombinant, since it contains restriction endonuclease cleavage sites characteristic of both parents, the endogenous resident SV40 genome of the transformed monkey cells and the exogenous tsD202 mutant.     

Functional rescue of temperature-sensitive defects in the cell-cycle mutants of rat 3Y1 fibroblasts by the small t-antigen deletion mutant of simian virus 40

We examined the effects of large T antigen of simian virus 40 (SV40) on the proliferation phenotypes of temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which cease proliferating in the G1 phase of the cell cycle at a restrictive temperature (39.8 degrees C). Four ts mutants, each representing independent complementation groups, were transformed with the dl-884 mutant of SV40 which lacks the unique coding region for small t antigen. In the case of two ts mutants, their transformed derivatives did not cease proliferation at 39.8 degrees C. In the other two mutants, the transformed cells continued to enter the S phase but the cells became detached from the dishes thereafter, at 39.8 degrees C. The proliferation phenotypes of the dl-884-transformed cells at 39.8 degrees C were quite similar with those of the same mutants transformed with the wild-type SV40. These results indicate that large T antigen alone is sufficient to overcome the inhibition of cellular entry into S phase caused by four different ts defects and determines the proliferation phenotypes of the cells after entering the S phase at a restrictive temperature, and that small t antigen does not alter the cellular phenotypes determined by large T antigen.