Response of Simian Virus 40-Transformed Cell Lines and Cell Hybrids to Superinfection with Simian Virus 40 and Its Deoxyribonucleic Acid

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.     

Chromosome analyses and anchorage-independent growth of SV40-induced morphologically transformed epithelial cells from amniotic fluids

Epithelial cells from amniotic fluid cell cultures are morphologically transformed by simian virus 40, 20 to 30 d after infection. The cells of the transformed colonies are highly basophilic, have a high nuclear-to-cytoplasmic ratio, and show a dense growth pattern. The cells are virus producers, and ultimately, after continuous passage, the cell lines reach a crisis situation with no growth. Twelve morphologically transformed cell colonies were isolated from five different individuals for chromosome analyses after approximately 18 population doublings (second bottle passage). For all cell lines diploid cells were observed. Banding of the chromosomes revealed normal morphology of euchromatic and heterochromatic regions. The suggestion is made that chromosome alteration is not necessary, nor a prerequisite, for the morphologically transformed phenotype to be expressed and that the transformation process per se causes chromosomal instability. Tests for colony formation of the 12 cell lines in semisolid medium showed that different transformed colony isolates from the same individual donor of the cells either formed or did not form colonies in agar. The size of the colonies was also consistent within individuals as compared to between individuals. These limited results are suggestive of a dependence upon the genetic constitution of the individual donor of the cells for colony formation in soft agar.     

Chromosome analyses and anchorage-independent growth of SV40-induced morphologically transformed epithelial cells from amniotic fluids

Summary Epithelial cells from amniotic fluid cell cultures are morphologically transformed by simian virus 40, 20 to 30 d after infection. The cells of the transformed colonies are highly basophilic, have a high nuclear-to-cytoplasmic ratio, and show a dense growth pattern. The cells are virus producers, and ultimately, after continuous passage, the cell lines reach a crisis situation with no growth. Twelve morphologically transformed cell colonies were isolated from five different individuals for chromosome analyses after approximately 18 population doublings (second bottle passage). For all cell lines diploid cells were observed. Banding of the chromosomes revealed normal morphology of euchromatic and heterochromatic regions. The suggestion is made that chromosome alteration is not necessary, nor a prerequisite, for the morphologically transformed phenotype to be expressed and that the transformation process per se causes chromosomal instability. Tests for colony formation of the 12 cell lines in semisolid medium showed that different transformed colony isolates from the same individual donor of the cells either formed or did not form colonies in agar. The size of the colonies was also consistent within individuals as compared to between individuals. These limited results are suggestive of a dependence upon the genetic constitution of the individual donor of the cells for colony formation in soft agar. Supported by National Science Foundation Grant PCM77-15876.     

Replication of Simian Virus 40 Deoxyribonucleic Acid: Analysis of the One-Step Growth Cycle

The time course of replication of simian virus 40 deoxyribonucleic acid (DNA) was investigated in growing monolayer cultures of subcloned CV1 cells. At multiplicities of infection of 30 to 60 plaque-forming units (PFU)/cell, first progeny DNA molecules (component 1) were detected by 10 hr after infection. During the following 10 to 12 hr, accumulation of virus DNA proceeded at ever increasing rates, albeit in a non-exponential fashion. The rate of synthesis then remained constant, until approximately the 40th hour postinfection, when DNA replication stopped. Under these conditions, the duration of the virus growth cycle was approximately 50 hr. The time needed for the synthesis of one DNA molecule was found to be approximately 15 min. At multiplicities of infection of 1 or less than 1 PFU/cell, the onset of the linear phase of DNA accumulation was delayed, but the final rate of DNA synthesis was the same, independent of the input multiplicity. This was taken as a proof that templates for the synthesis of viral DNA multiply in the cell during the early phase of replication. However, the probability for every replicated DNA molecule to become in turn replicative decreased constantly during that phase. This could be accounted for by assuming a limited number of replication sites in the infected cell.     

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.     

Relationship Between Virus-Induced Cellular Deoxyribonucleic Acid Synthesis and Transformation by Simian Virus 40

The fraction of cells in a confluent 3T3 cell monolayer induced by simian virus 40 infection to replicate deoxyribonucleic acid and divide corresponds to those cells which eventually become transformed. Virus-induced cells were partially separated from noninduced cells by sedimentation through Ficoll gradients. Three- to eightfold higher transformation frequencies were obtained with those cells that began to synthesize cellular deoxyribonucleic acid and divide shortly after simian virus 40 infection as compared to noninduced cells.     

Physiological State of Human Embryonic Lung Cells Affects Their Response to Human Cytomegalovirus

Cultures of human embryonic lung (HEL) cells in different physiological states were studied for their susceptibility to infection with human cytomegalovirus (CMV) with respect to production of infectious virus, synthesis of viral antigens, and virus-induced stimulation of cellular DNA synthesis. In general, subconfluent, actively growing cells yielded higher amounts of infectious virus than did confluent contact-inhibited cells. The higher yield of infectious virus was correlated with a greater percentage of cells producing viral antigens within the first 48 h after infection. In confluent cultures, 25 to 50% of the cells produced viral antigens within the first 48 h postinfection. This proportion did not change over a 10-fold range of multiplicity of infection, indicating that many of the cells in confluent cultures did not support productive infection. However, virtually all the cells in subconfluent cultures were susceptible. Also, in contrast to herpes simplex virus and pseudorabies virus, infectious CMV is not produced by cells treated with 5-fluorouracil and thymidine. Virus-induced stimulation of cellular DNA synthesis in cells infected at high multiplicities of infection could be detected only in confluent cultures, in which cellular DNA synthesis had been previously suppressed, but could not be detected in similarly treated cultures of subconfluent cells. The lack of detectable stimulation of cellular DNA synthesis in the latter was related to the fact that practically all the cells in the culture synthesized viral antigens within the first 48 h after infection, productive infection and detectable synthesis of cellular DNA being mutually exclusive.     

Superinfection of Simian Virus 40-Transformed Permissive Cells with Simian Virus 40

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : I. Biologic, Virologic, and Chemical Properties

Two contact-inhibited "revertant" cell lines were isolated from an SV40-transformed mouse 3T3 cell line (SV-3T3) after exposure to 5-fluoro-2'-deoxyuridine. Revertant cells resembled 3T3 cells morphologically and grew to saturation densities which were similar to those of 3T3 cells; however, revertant cells readily formed both single and multinucleated giant cells in confluent cultures. SV40 virus was rescued from revertant cells by fusion with permissive monkey cells. The rescued virus transformed 3T3 cells with the same efficiency as wild type virus, and produced transformed colonies which were phenotypically similar to those produced by wild type virus. The revertant cells also resembled normal 3T3 cells in that they contained higher quantities of sialic acid than SV-3T3 cells. An inverse correlation was found between the saturation density of cells and their sialic acid content. Collagen content, however, of revertant cells was similar to that of SV-3T3 cells. The data presented suggest that the property of contact inhibition in revertant cells is related to the sialic acid content of the plasma membrane and that changes in sialic acid content of transformed cells are not directly specified by the viral genome.     

Morphological Aspects of the Uptake of Simian Virus 40 by Permissive Cells

After exposure of permissive cells to simian virus 40 (SV40), single particles were engulfed by the cell membrane and transported to the nucleus. The cell membrane closed tightly around the particles, increasing their diameter from 40 to 55 nm. The cell membrane was lost during interaction with the nuclear membranes, and particles of the original size were found in the nucleus 1 hr after infection. Uncoating of these nuclear particles occurred rapidly, and none could be found 4 hr after infection. Viral progeny appeared 24 hr after infection.     

Cell killing by Frog Virus 3: Evidence for cell killing by single viral particles or single viral subunits

Cell killing by Frog Virus 3 was assayed after infection of chinese hamster ovary cells under non permissive conditions for virus multiplication. The kinetics of the loss in the efficiency of colony formation as a function of the virus multiplicity indicated that infection of a cell with a single viral particle brought about cell death. About 15 percent of the cells exhibited transient resistance to killing by single viral particles. Treatment of cells with proteins solubilized from Frog Virus 3 also resulted in cell killing with one hit kinetics thus implying that the interaction with a single viral subunit sufficed to entail cell death.     

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.     

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.     

Thymidine Kinase from Normal, Simian Virus 40-Transformed and Simian Virus 40-Lytically Infected Cells

Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.     

Simian Virus 40-Host Cell Interaction During Lytic Infection

Both exponentially growing and serum-arrested subcloned CV-1 cell cultures were infected with simian virus 40 (SV40). By 24 h after infection 96% of the nuclei of these permissive cells contained SV40 T-antigen. Analysis of the average DNA content per cell at various times after infection indicated that by 24 h most of the cells contained amounts of DNA similar to those normally found in G(2) cells. Analysis of cell cycle distributions indicated that a G(2) DNA complement was maintained by over 90% of the cells in the infected populations 24 to 48 h postinfection. Cells continued to synthesize SV40 DNA during the first 50 h after infection, and cytopathic effect was first observed 60 h after inoculation. After infection the number of mitotic cells that could be recovered by selective detachment decreased precipitously and was drastically reduced by 24 h. A study of the kinetics of decline in the number of mitotic cells suggests that this decline is related to an event during the cell cycle at or near the G(1)-S-phase border upon which commencement of SV40 DNA replication apparently depends. It was concluded that after SV40 infection, stationary cells are induced to cycle, and cycling cells complete one round of cellular DNA synthesis but do not divide. Although the infected cells continue to synthesize viral DNA, they do not appear able to reinitiate cellular DNA replication units. These results imply that the abundance of T-antigen (produced independently of cell cycle phase) in the presence of the enzymes required for continued DNA synthesis is not sufficient for reinitiation of cellular DNA synthesis.     

Antigenic properties and cellular localization of herpes simplex virus glycoprotein H synthesized in a mammalian cell expression system.

Herpes simplex virus type 1 glycoprotein H (HSV-1 gH) was synthesized in an inducible mammalian cell expression system, and its properties were examined. The gH coding sequence, together with the stable 5' untranslated leader sequence from xenopus beta-globin, was placed under control of the strong promoter from the human cytomegalovirus major immediate-early gene in an amplifiable plasmid which contains the simian virus 40 (SV40) virus origin for replication (ori). This expression vector was transfected into ts COS cells constitutively expressing a temperature-sensitive SV40 T antigen which allows utilization of the SV40 ori at permissive temperatures. The results of transient expression assays at the permissive temperature showed that HSV-1 gH could be synthesized in greater amounts than those produced by a high-multiplicity virus infection. The proteins produced were detected in Western blots (immunoblots) with a HSV-1 gH-specific polyclonal serum raised against a TrpE-gH fusion protein. The transfected gH had an apparent molecular weight of approximately 105,000, intermediate in size to those of the precursor (100,000) and fully processed forms (110,000) of HSV-1 gH from infections. Antigenicity was investigated by reactions with three virus-neutralizing monoclonal antibodies specific for conformational epitopes on gH. Only one of these monoclonal antibodies could immunoprecipitate the synthesized gH. However, equal recognition of the transfected gH was achieved by superinfection with virus. In addition, detectable amounts of gH were not expressed on the cell surface unless the cells were superinfected with virus. Studies with a temperature-sensitive mutant, ts1201, defective in encapsidation showed that the changes in antigenic structure and cell surface expression caused by superinfection with virus were not due simply to incorporation of gH into virions. These results suggest that gH requires additional virus gene products for cell surface localization and formation of an antigenic structure important for its function in mediating infectivity.     

Growth state of the cell early after infection with simian virus 40 determines whether the maintenance of transformation will be A-gene dependent or independent.

The existence of both temperature-sensitive (N) and temperature-insensitive (A) rat transformants, isolated after infection with simian virus 40 tsA mutant, is reported. Both types can be isolated as dense foci. Foci appearing after infection of rapidly growing cells were temperature sensitive. Infection of cells arrested at confluence gave rise to foci that were temperature insensitive. Transformants isolated by the agar assay (conditions under which normal cells are unable to grow) were also temperature-insensitive. N-transformants remained temperature sensitive upon entering the resting state at the restrictive temperature and upon re-entering the growth cycle at the permissive temperature. They also remained temperature sensitive under a variety of conditions restrictive for nontransformed cells. Thus, the state of the cell in the first few days after infection fixes the cells. Thus, the state of the cell in the first few days after infection fixes the cell as an N- or A-transformant. Various models for transformation are discussed, including one proposing that the virus interacts in two ways with a central cell mechanism controlling growth. The maintenance of the transformed phenotype would be dependent on T-antigen in N-transformants but independent of T-antigen in A-transformants.