Rescue of defective SV40 from mouse-human hybrid cells containing human chromosome 7

Subcloning of SV40 T-antigen positive, V-antigen negative mouse human hybrid clones resulted in their segregation into T-antigen positive and negative subclones which still contained the full complement of mouse chromosomes. These subclones were analyzed for SV40 V-antigen and virion formation after fusion of the subclone cells with CV-1 cells of African green monkey kidney origin. All subclones which contained the human chromosome 7 showed the appearance of V-antigen in heterokaryocytes after fusion with CV-1 cells; all subclones, which have lost the human chromosome 7, did not express V-antigen following fusion with CV-1 cells. Wild-type plaque-forming SV40 could not be rescued from cultures of any of the T-antigen positive or negative clones or subclones, but defective SV40 virions were rescued from a hybrid subclone in which the only human chromosome present was the 7; two subclones which had lost human chromosome 7 showed no SV40 virion formation.     

Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line

We are exploring the origin and function of amplified DNA sequences associated with double minutes (DMs) in a spontaneously transformed derivative of mouse 3T3 cells. Toward that goal, we have constructed a cDNA library using RNA from these cells and have isolated cDNA clones representing sequences that are amplified and overexpressed in these 3T3-DM cells. From results of Northern- and Southern-blot analyses, we conclude that these cDNAs represent two distinct genes, which we have designated mdm-1and mdm-2.Using DNAs from a panel of Chinese hamster-mouse somatic cell hybrids together with in situ hybridization protocols for gene mapping studies, we have found that these DM-associated, amplified DNA sequences originate from mouse chromosome 10, region C1–C3. Sequences homologous to mdm-1and mdm-2are present in the genomes of several species examined, including that of man.     

Characterization of defective SV40 isolated from SV40-transformed cells.

Defective SV40 viruses were isolated from SV40-transformed monkey, human and hamster cells after Sendai virus-mediated fusion of the transformed cells with TC7 cells, a stable line of African green monkey kidney cells. Viral isolates were concentrated and purified and the defective viruses examined by electron microscopy. The buoyant densities in CsCl of the defective viruses ranged between 1.32 and 1.33 g/cc. DNA isolated from defective viruses was characterized by dye-buoyant density centrifugation and by velocity sedimentation in neutral CsCl. The DNA was heterogeneous in size and contained some covalently closed double-stranded circular molecules.     

Rescue of SV 40 from interferon-treated heterokaryons

Summary Simian virus 40 (SV 40)-transformed nonpermissive cells express only the early products of SV 40. Heterokaryons formed by fusion of these transformed cells with uninfected permissive cells support the activation of the resident viral genome leading to subsequent viral DNA replication, late protein synthesis and release of progeny virus. Pretreatment of heterokaryon cultures with either mouse or monkey interferon (IFN) before fusion with polyethylene glycol (PEG) produced a dose-dependent inhibition in the appearance of free viral DNA as well as production of infectious virus. The decreased yield of SV 40 in these cultures was similar to the inhibition which was observed in mouse or monkey cells incubated with homologous IFN prior to exogenous infection with SV 40. when IFN was added to the cultures at progressively later times after fusion with PEG, there was less inhibition of virus production. Although there was a comparable decrease in the production of virus by pretreatment with either mouse or monkey IFN, monkey IFN exerted the inhibition for a longer period of time when added after heterokaryon formation. These results demonstrate that IFN treatment applied even after initiation of SV 40 replication can still inhibit virus multiplication.With 1 Figure     

T antigen and initiation of cell DNA synthesis in a temperature-sensitive mouse line transformed by an SV40ts a mutant and in heterokaryons of the transformed cells and chick erythrocytes

The role of SV40 gene A product in initiation of cellular DNA synthesis was investigated, using a mouse kidney line [mKSA207] transformed by SV40ts A207. mKSA207 cells were temperature sensitive for growth, lost SV40 T antigen (Tag) when incubated in low serum at 40°C, and accumulated Tag in the cytoplasm when fed 10% serum and incubated at the nonpermissive temperature (39.7°C). Following serum addition, the percentage of mKSA207 cells synthesizing DNA was essentially the same at nonpermissive (39.7°C) and permissive temperatures (33.5°C). The cells entered S phase asynchronously at both temperatures, but most cells entered S within 16 h, and before Tag accumulated. mKSA207 synchronized by a double thymidine block also synthesized DNA at 39.7°C and entered a second S phase. Tag-depleted or Tag-synchronized mKSA207, when fused with chick erythrocytes (CE), activated CE DNA synthesis. At nonpermissive temperatures (39.7°C), 40% of CE nuclei in heterokaryons with Tag-depleted mKSA207 displayed³H-thymidine-labeled nuclei 28–40 h after fusion, when only 12% of CE nuclei were Tag⁺. The experiments indicate that SV40 gene A product probably does not have a direct role as initiator of cellular DNA synthesis.A preliminary report of these findings was presented at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, Louisiana, May 8–13, 1977.     

Establishment of transformed swine fibroblast cell lines using SV40 large T antigen

Summary Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.     

Analysis of the nonviral antigens immunoprecipitable by SV40 T antibody from SV40-transformed human/mouse hybrid cell lines

A temperature-sensitive (ts) mutant of herpes simplex virus type 1 (HSV-1), tsJ12, is able to undergo one cycle of replication at the nonpermissive temperature (39°) yielding wild-type quantities of enveloped virus particles. These particles contain viral DNA which is as infectious as wild-type viral DNA; however, they are not infectious. Analysis of [¹⁴C]glucosamine-labeled mutant-infected cell extracts by one- and two-dimensional polyacrylamide gel electrophoresis demonstrated that at 39° tsJ12 fails to induce the synthesis of both the mature gB glycoprotein and its dimeric form which are normal constituents of the virion envelope. Polyethylene glycol, an agent which promotes membrane fusion, enhances the infectivity of tsJ12 virions by greater than 1000-fold following adsorption of virus to susceptible cells demonstrating that mutant virions are able to attach to cells but not penetrate. Consistent with a defect in the virion envelope, tsJ12 is able to interfere with the production of infectious wild-type virus, presumably by the formation of pseudotypic virions composed of wild-type viral genomes in gB-deficient envelopes. Physical mapping of the is defect in this mutant demonstrates that it lies within the limits of the DNA sequence which specifies gB on the physical map of the genome. A ts⁺ revertant of tsJ12 is as infectious as wild-type virus and synthesizes a gB glycoprotein which is indistinguishable from that of wild-type virus. Thus, biological and biochemical studies of tsJ12 and of a ts+ revertant of this mutant (1) demonstrate that glycoprotein gB is essential for infectivity at the level of penetration and (2) further define the physical map location of the gene for this glycoprotein.     

Properties of an SV40 transformed African green monkey kidney (BSC-1) cell line: interactions between SV40 and cellular DNA metabolism

Interactions between SV40 and cellular DNA metabolism have been studied in BSC-1 cells and in a clonal line derived from BSC-1 cells transformed by SV40 ($\text{BSC}\text{SV Cl}\text{.1.1}$) which is still susceptible to superinfection by SV40 virus. In BSC-1 cells, under certain conditions, the virus stimulates the synthesis of high molecular weight DNA, releases the inhibition of replication caused by bromodeoxyuridine and triggers the production of low molecular weight cellular DNA. These interactions occur in uninfected BSC/SV cells and are unaffected by superinfection, suggesting that viral functions involved in the control of cellular DNA metabolism are continuously expressed in these cells.     

A nonselective analysis of SV40 transformation of mouse 3T3 cells

Mouse cells transformed by simian virus 40 show many alterations in their growth properties in vitro. In order to investigate the coordinate nature of these changes, we have analyzed the growth properties of 40 randomly selected colonies arising after SV40 infection of 3T3 cells. Clones of cells, established from these colonies, were characterized as to saturation density and doubling time in 10% and 1% calf serum, growth in methyl cellulose suspension, colony formation on monolayers of normal cells, and presence of viral antigens. This analysis revealed that only 5 of the clones were indistinguishable from 3T3 cells; the remaining 35 clones differed from 3T3 cells in that they grew as rapidly in 1% calf serum as standard SV40 transformed cells. Of these 35 clones, ten corresponded to standard transformants previously described. Another ten showed other growth properties intermediate between 3T3 cells and standard transformants. These intermediate clones had lower levels of viral T-antigen than standard transformants and showed considerable heterogeneity in staining from cell to cell. The remaining 15 clones were T-antigen negative and had saturation densities slightly higher than that of 3T3 cells. These changes in cellular behavior are stable on recloning.     

Reversion to normal phenotype induced by SV40 in a spontaneously transformed malignant Chinese hamster cell line

By using a selection procedure that excluded the transforming effect of SV40, reversions to several properties of normal phenotype were for the first time obtained in a transformed Chinese hamster cell line after SV40 infection. The value of induction to recovery of contact inhibition was typical for SV40-induced reverse gene mutations. Thirteen of 15 isolated revertant clones were T-antigen positive, thus synthesizing the product of viral oncogene. Therefore, in the majority of clones reversion occurred in spite of the presence of viral transforming protein. Dot hybridization revealed the presence of SV40 DNA in all revertants including those expressing no T antigen. The virus rescued from one T-antigen positive and two negative clones proved to be infectious. Reversion to contact inhibition was followed by reversion as regards serum requirements and growth in soft agar. However, in all cases reversion was partial. Karyologic analysis of revertant clones showed that six clones maintained the hypodiploid karyotype of the parental clone, six revertants were near-tetraploid, and one was near triploid. The possible events underlying the SV40-induced reversions to normal phenotype and the role of virus-induced mutations in viral carcinogenesis are discussed.     

Herpes simplex virus-induced "rolling circle" amplification of SV40 DNA sequences in a transformed hamster cell line correlates with tandem integration of the SV40 genome

Infection with herpes simplex virus leads to amplification of SV40 DNA in various SV40-transformed cells. In earlier studies with the SV40-transformed hamster cell line Elona two different types of DNA amplification could be identified: (i) Bidirectional overreplication of chromosomally integrated SV40 DNA expanding into the flanking cellular sequences ("onion skin" type) and (ii) highly efficient synthesis of extremely large head-to-tail concatemers containing exclusively SV40 DNA ("rolling circle" type). These investigations have indicated that the chromosomally integrated form of SV40 might be the substrate for both types of overreplication. There still had been uncertainties as to whether and how these events were connected. A hypothetical assumption of a recombinational event leading to the excision of SV40 DNA molecules is supported by the results presented here: In this study cloned Elona cell lines were investigated for their ability to amplify SV40 sequences and for the mechanism of amplification utilized. SV40 integration in a partial tandem manner correlates with a strong rolling circle amplification. In contrast, in one cell line harboring a truncated SV40 genome, amplification appears mainly restricted to intrachromosomal bidirectional overreplication. Possible implications for HSV functions involved in the amplification process will be discussed.     

Monoclonal antibody to SV40 T-antigen blocks lysis of cloned cytotoxic T-cell line specific for SV40 TASA

The lysis of SV40-transformed target cells by a cloned cytotoxic T cell line (CTB6) that is SV40 TASA-specific and H-2Kb-restricted is blocked by a monoclonal antibody reactive with the SV40 T-antigen. The blocking is maximized at high antibody concentrations and low effector-to-target cell ratios. These data indicate that at least one antigenic determinant of the SV40 T-antigen molecule is expressed at the cell surface and is either recognized by this cytotoxic T lymphocyte or is proximate to that determinant recognized by this cytotoxic T lymphocyte.     

SV40-transformed cells express SV40 T antigen-related antigens on the cell surface

SV40 tumor antigen (T-Ag)-related antigens were detected serologically on the surface (surface T) of living SV40-transformed human and mouse monolayer cells by an ¹²⁵I-protein A binding assay. In immunofluorescence analysis, these cells were negative for surface T. However, on mKSA, a SV40-transformed mouse cell line grown in suspension or on SV40-transformed human and mouse monolayer cells put into suspension, surface T could be visualized by immunofluorescence microscopy. The antisera used in these experiments were raised in rabbits with purified, SDS-denatured SV40 T-Ag or came from hamsters bearing SV40 tumors. Both types of antisera had in common high titers against SV40 T-Ag (?1:1000). All these antisera were negative on normal cells or on polyoma virus-transformed cells. The specificity of both antisera for SV40 T-Ag-related binding sites on the surface of SV40-transformed cells were demonstrated by an ¹²⁵I-IgG blocking assay in which preincubation of the cells with rabbit anti-T-Ag serum inhibited the binding of hamster SV40 tumor serum to the cell surface by about 85%. These results demonstrate the expression of T-Ag-related antigens on the surface of living cells and, therefore, support the hypothesis that SV40 T-Ag-related antigens participate in the formation of the SV40-specific tumor transplantation antigen (TSTA).