Characterization of simian cells tranformed by temperature-sensitive mutants of simian virus 40.

Seven lines derived from primary African green monkey kidney cells, which had survived lytic infection by wild-type simian virus 40 (SV40) or temperature-sensitive mutants belonging to the A and B complementation groups, were established. These cultures synthesize SV40 tumor (T) antigen constitutively and have been passaged more than 60 times in vitro. The cells released small amounts of virus even at high passage levels but eventually became negative for the spontaneous release of virus. Virus rescued from such "nonproducer" cells by the transfection technique exhibited the growth properties of the original inoculum virus. Four of the cell lines were tested for the presence of altered growth patterns commonly associated with SV40-induced transformation. Although each of the cell lines was greater than 99% positive for T antigen, none of the cultures could be distinguished from primary or stable lines of normal simian cells on the basis of morphology, saturation density in high or low serum concentrations, colony formation on plastic or in soft agar, hexose transport, or concanavalin A agglutinability. However, the cells could be distinguished from the parental green monkey kidney cells by a prolonged life span, the presence of T antigen, a resistance to the replication of superinfecting SV40 virus or SV40 viral DNA, and, with three of the four lines, an ability to complement the growth of human adenovirus type 7. These properties were expressed independent of the temperature of incubation. These results indicate that the presence of an immunologically reactive SV40 T antigen is not sufficient to ensure induction of phenotypic transformation and suggest that a specific interaction between viral and cellular genes and/or gene products may be a necessary requirement.     

Products of complementation between temperature-sensitive mutants of simian virus 40.

Temperature-sensitive mutants of the D complementation group of simian virus 40 exhibit delayed complementation. Analysis of the thermal stability, kinetic profiles in temperature shift experiments, and progeny of complementation have led to the hypothesis that delayed complementation is not true complementation, but the result of a very low level of leakiness, followed by phenotypic mixing of the progeny D mutants. This hypothesis is consistent with the proposal that D mutants are defective in uncoating. In the course of these experiments, it was observed that fresh medium suppresses the growth of D mutants at the restrictive temperature.     

DNA sequence alterations responsible for the synthesis of thermosensitive VP1 in temperature-sensitive BC mutants of simian virus 40.

The segment of simian virus 40 (SV40) genome which is recognized as the BC domain encodes for the COOH-terminal end of the SV40 major capsid protein VP1. Mutations in this domain lead to the synthesis of a thermosensitive VP1 which fails to assemble mature SV40 at the nonpermissive temperature. We determined the DNA sequences of eight BC mutants and compared them with the DNA sequences of wild-type SV40, polyomavirus, and BK virus. We found that BC11 and BC223 mutations result from changes in nucleotide residues 2367 (A to C) and 2084 (C to T), respectively. The others (i.e., BC208, BC214, BC216, BC217, BC248, and BC274) share the same point mutation at nucleotide 2354 (C to T). These mutations resulted in the following changes: Lys to Thr, His to Tyr, and Pro to Ser at VP1 amino acid residues 290, 196, and 286, respectively.     

Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.

We investigated the molecular properties of eight temperature-sensitive mutants of simian virus 40 large T antigen (tsA mutants). The mutants have single amino acid substitutions that block DNA replication at 39 to 41 degrees C in vivo. In vitro, five of the mutant proteins were highly sensitive to a brief heat shock at 39 degrees C, while the three remaining proteins were only partially sensitive at 41 degrees C. We characterized the five most defective mutant proteins, using a variety of biochemical assays for replication functions of T antigen. Heat shock of purified T antigen with a mutation at amino acid 422 significantly impaired the oligomerization, origin-binding, origin-unwinding, ATPase, and helicase functions of T antigen. In contrast, substitution of amino acid 186, 357, 427, or 438 had more selective, temperature-sensitive effects on T-antigen functions. Our findings are consistent with the conclusion that T antigen functions via a hierarchy of interrelated domains. Only the ATPase activity remained intact in the absence of all other functions. Hexamer formation appears to be necessary for core origin-unwinding and helicase activities; the helicase function also requires ATPase activity. All five tsA mutants were impaired in functions important for the initiation of DNA replication, but three mutants retained significant elongation functions.     

DNA replication and chromatin structure of simian virus 40 insertion mutants.

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.     

Excision of viral DNA from host cell DNA after induction of simian virus 40-transformed hamster cells.

Simian virus 40-transformed hamster cells were induced to produce infectious virus by treatment with mitomycin C or gamma-irradiation. A portion of the simian virus-40 DNA, which is integrated into the host cell genome in uninduced cells, was recovered in a pool of relatively low-molecular-weight DNA early after induction treatment in the absence of DNA replication. The data indicte that excision of the viral genome occurs subsequent to the induction stimulus.     

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.     

Salt-stable association of simian virus 40 capsid with simian virus 40 DNA

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.     

Reinitiation of host DNA synthesis in senescent human diploid cells by infection with simian virus 40

Human diploid fibroblasts, TIG-1, cease to proliferate at about 60-62 population doubling level. In their senescent state used in this study, the percentage of nuclei labeled by [3H]thymidine for 48 h was around 1-2% in fresh medium containing 5-40% fetal bovine serum. The percentage of labelled nuclei increased up to 10-fold after infection with SV40. This increase reflects stimulation of cell DNA synthesis because: 1. The increase also occurred when ts A900 was used for infection at the non-permissive temperature, under these conditions viral DNA synthesis is inhibited; 2, the increase paralleled the stimulation of [3H]thymidine incorporation into DNA in a Hirt-precipitate fraction from SV40-infected cells. UV-irradiated SV40 had reduced ability to induce DNA synthesis. A viable deletion mutant of SV40, d1940, had almost the same activity to induce cell DNA synthesis as did wild-type SV40. Equilibrium density gradient centrifugation analysis of DNA labelled with 5-bromodeoxyuridine (BrdU) supported semiconservative replication rather than repair synthesis. We conclude that a considerable fraction of human diploid cells in a senescent population initiate host DNA replication by infection with SV40, although these cells cannot be stimulated with fetal bovine serum.     

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.     

Porcine endometrial epithelial cells immortalized by transfection with origin-defective, temperature-sensitive simian virus 40 DNA.

The purpose of this study was to immortalize porcine endometrial cells and to characterize the transformed cells. Primary porcine endometrial cells were transfected with the plasmid vector (pmk16) containing SV40 DNA using a liposome-mediated method. The viral DNA was from a replication-defective, origin-minus, temperature-sensitive mutant strain (A58). One clone, designated PE-1, has been propagated for over 120 passages. PE-1 cells grown at 33C (33C cells) exhibit spindle-shaped morphology; when cultured at 40C (40C cells), they took on a polygonal or spherical shape. Morphology of 40C cells returned to the spindle shape after culture flasks were shifted back to 33C. During a 2-week period, 33C cells propagated approximately 30-fold faster than 40C cells, whereas protein concentration was higher in 40C cells. Southern blot analysis of PE-1 cells demonstrated successful integration of the ts-SV40 DNA sequence into the porcine endometrial cells, possibly at multiple sites. The presence of cytokeratin on PE-1 cell membranes was shown by immunocytochemical studies, suggesting that the PE-1 cell clone was of epithelial origin. Reverse phase (RP)-HPLC analysis of PE-1 cell extract indicated that the majority of immunoreactive beta-endorphin (ir-BEND) eluted with a hydrophobicity similar to that of synthetic BEND and alpha-N-acetylated BEND (Nac-BEND). These results demonstrate that a porcine endometrial cell line has been established, and that this cell line possesses characteristics of temperature sensitivity in cell morphology, growth rate, and protein synthesis.     

Regulatory mutants of simian virus 40: constructed mutants with base substitutions at the origin of DNA replication.

Mutants of simian virus 40 (SV40) with base substitutions at or near the origin of replication of the viral genome have been constructed by bisulfite mutagenesis at the BglI restriction site of SV40 DNA, followed by transfection of cells with the BglI-resistant (BglI^r) DNA so generated. Based on plaque morphology at different temperatures, the resulting BglI^r mutants could be classified into four-groups. Class I mutants (designated ar for “altered restriction”) were indistinguishable from wild-type SV40; class II mutants (designated shp for “sharp plaque”) produced small, sharp-edged plaques; class III mutants (designated sp for “small plaque”) produced small plaques at 32 °C, 37 °C and 40 °C; and class IV mutants (designated cs for “cold sensitive”) produced small plaques at 32 °C and wild-type plaques at 37 °C and 40 °C. That the altered plaque morphology of sp and cs mutants was related to mutation at the BglI restriction site was demonstrated by co-reversion to wild-type of the plaque phenotype and BglI sensitivity. The nucleotide sequence around the original BglI site was determined in the DNA from one mutant of each class. In each case a different base-pair substitution was found, at a site outside sequences coding for SV40 proteins. When rates of replication of mutant DNAs were measured during productive infection, ar mutant DNA was synthesized at a rate comparable to that of wild-type SV40 DNA, shp mutant DNA was made at a rate exceeding that of wild-type, sp mutant DNA was synthesized at a lower rate than that of wild type. and cs mutant DNA synthesis was reduced at 32 °C, but about the same as the wild-type rate at 40 °C. These patterns of mutant DNA synthesis were unaltered in cells co-infected with mutant and wild-type virus, i.e. the defects in DNA synthesis were not trans-complementable. We conclude that the defective mutants have single base-pair changes in a cis element that determines the rate of viral DNA replication, presumably within the origin signal itself.     

New host cell system for regulated simian virus 40 DNA replication.

Transformed monkey cell lines (CMT and BMT) that inducible express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitation of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of synthesis on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to fourfold more total T antigen after 2 days of induction by comparison with uninduced controls. A direct correlation was found between the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells. Inducible BMT cells expressing a low basal level of T antigen were efficiently transformed by a vector carrying the neomycin resistance marker and an SV40 origin of replication. These vector sequences were maintained in an episomal form in most G418-resistant cell lines examined and persisted even in the absence of biochemical selection. Extensive rearrangements were observed only if the vector contained bacterial plasmid sequences. Expression of a protein product under the control of the SV40 late promoter in such vectors was increased after heavy-metal-dependent amplification of the template. These results demonstrate the ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state.     

DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies.     

Simian virus 40 mutants carrying two temperature-sensitive mutations.

Five temperature-sensitive mutants of simian virus 40 containing two temperature-sensitive mutations were isolated. The double mutant of the A and D complementation groups, like the D mutants, failed to complement by conventional complementation analysis and did not induce host DNA synthesis at 40 degrees C. However, under conditions that suppressed the D defect, the A:D double mutant expressed only the A defect. Thus, viral DNA replication dropped rapidly after this mutant was shifted from permissive to restrictive temperatures. The A:D double mutant failed to transfrom at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. Double mutants of A:B, A:C, and A:BC complementation groups, like their A parent, were defective in viral DNA replication, in the induction of host DNA synthesis and in the transformation of secondary Chinese hamster lung cells at the nonpermissive temperature.     

Perturbations in simian virus 40 DNA synthesis by ultraviolet light.

Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive monkey CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e. completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.     

DNA synthesis by partially purified replicating simian virus 40 chromosomes.

We have partially purified replicating simian virus 40 (SV40) chromosomes in a form which allows continued DNA synthesis in vitro. We first prepare a soluble DNA-synthesizing system from SV40-infected monkey cells and then sediment the components through a neutral sucrose gradient of extremely low ionic strength. Replicating SV40 chromosomes isolated from such gradients are capable of continuing DNA synthesis in vitro in the same manner as two crude subnuclear systems we have previously described (4). This indicates that the enzymes and other proteins required for in vitro DNA synthesis are bound to the replicating chromosomes.     

Integration of progeny simian virus 40 DNA into the host cell genome

A procedure was developed for the separation of cellular DNA of productively infected monkey kidney cells from free simian virus 40 DNA. The application of this procedure allowed the investigation of progeny viral DNA integration into the host cell DNA by nucleic acid hybridization techniques. The purification consisted of precipitation of the cellular DNA by Hirt's (1967) method, velocity centrifugation in alkaline sucrose gradients, equilibrium centrifugation in ethidium bromide/CsCl solution, and an additional velocity centrifugation in an alkaline sucrose gradient. The efficiency of each step of the procedure was determined by monitoring the amount of contaminating free viral DNA. Purified cellular DNA, isolated from cells late after infection, contained approximately 0/sd006% free viral DNA, but as much as 2% integrated simian virus 40 DNA. This corresponds to more than 20,000 integrated virus genome equivalents per cell, as determined by DNA-DNA reassociation kinetics. Integration of simian virus 40 DNA into the cellular DNA became detectable at 24 hours after infection, and increased with the increase in the rate of viral DNA synthesis.     

Cold-sensitive regulatory mutants of simian virus 40

A preparation of short synthetic myosin filaments (minifilaments) in the absence of other myosin forms is reported. Myosin minifilaments have been prepared by dialysing myosin from vertebrate striated muscle into 10 mm-citrate/Tris buffer (pH 8.0 at 4 °C) containing no other salt. These polymers of myosin are very stable and show little tendency to aggregate or dissociate in the original solvent. Sedimentation velocity, diffusion and viscosity measurements indicate that the minifilaments are composed of 16 to 18 molecules. Examination of electron micrographs reveals that the bare central region of minifilaments extends over 1600 to 1800 Å and the entire particles are about 3000 Å long with a diameter of 80 Å across the smooth region. They have the appearance of short bipolar filaments (Huxley, 1963). In solution the minifilaments are homogeneous in terms of size distribution and exhibit normal MgATPase and CaATPase activities. When examined in the ultracentrifuge, the minifilaments sediment in the form of a hypersharp peak (or bar) with a sedimentation coefficient independent of rotor speed. The minifilaments can be dissociated by ATP, hardly by MgATP; whereas KCl (between 0.04 and 0.2 m) induces further polymerization. It is suggested that the minifilaments are an intermediate in the assembly of myosin filaments.