H-2Kb mutations limit the CTL response to SV40 TASA

The cytotoxic T lymphocyte (CTL) responses directed towards SV40 tumor-associated specific antigen (TASA) in nine strains of spontaneously arising Kb mutant mice were analyzed. All nine mutants generated normal levels of H-2Db-restricted response, but the K-end-restricted CTL response varied. B6.C-H-2bm1 (bm1) did not produce K-end-restricted SV40 TASA-specific CTL upon immunization, and SV40-transformed bm1 cells were not lysed by intra-H-2 recombinant Kb [B10.A(5R)] CTL. Nonreciprocal cross-reactive lysis was seen between B6-H-2bm8 (bm8) and B10.A(5R). Strain B6-H-2bm8 mice produce highly specific Kbm8-restricted CTL that lyse SV40-transformed bm8 cells (Kbm8SV) but not B10.A(5R) target cells (K5RSV), although Kbm8SV targets can be partially lysed by B10.A(5R) CTL. The other seven Kb mutants cross-react with B10.A(5R). These experiments definitively show that genes mapping to the K and/or D region directly control the H-2-restricted CTL response to SV40 TASA.     

H-2 antigen requirements in the in vitro induction of SV40-specific cytotoxic T lymphocytes

T cytotoxic cells generated to syngeneic SV40 virus transformants lyse only SV40 target cells that are syngeneic at the H-2 locus. In contrast, SV40-specific tumor transplantation immunity shows no requirements for syngeneic H-2. Inoculation of allogeneic or even xenogeneic transformants will confer immunity to a challenge of syngeneic SV40 tumor cells. The experiments described here represent an attempt to reconcile these apparently conflicting observations. In our hands, generation of SV40-specific T cytotoxic cells in vitro requires both in vivo priming and secondary in vitro sensitization. We have found that priming for a secondary syngeneic-restricted response requires only that the cell employed be SV40 transformed. That is, priming may be accomplished with syngeneic, allogeneic, or xenogeneic SV40 transformants. Thus, the apparent lack of H-2 restriction in vivo immunity does not eliminate a role for the H-2-restricted cytotoxic T cell in tumor transplantation immunity.     

Analysis of a defect in the H-2 genes of SV40 transformed C3H fibroblasts that do not express H-2Kk

C3H fibroblasts transformed in vitro with SV40 were adapted to in vivo growth. Several clones were isolated from a single, highly oncogenic tumor and those that displayed oncogenic potential also no longer expressed the H-2Kk molecule. Using the technique of Southern blot hybridization, the H-2 genes and integrated SV40 sequences present in the genomic DNA of several of these clones have been examined and compared with both the parent line and normal liver genomic DNA from C3H mice. All H-2Kk negative clones had altered H-2 genes that appeared as a gain and, depending on the restriction endonuclease, loss of hybridizing fragments compared to normal C3H DNA. A 5.5-kb fragment missing from the Sstl digests of the H-2Kk negative variants was mapped to the H-2Kk region of the major histocompatability complex with the use of congenic mice. This provided direct evidence that a mutation had occurred in the H-2Kk region. The integrated SV40 sequences were similar to those already seen in other SV40 transformed cells and not closely linked to any of the H-2 genes. There was no indication that the H-2 mutation was caused by integration of SV40.     

Purification of SV40 T-antigen by SV40 DNA-sepharose affinity chromatography

T-antigen from SV40-infected BSC-1 cells was purified approximately 30,000 fold using a rapid purification procedure consisting of ammonium sulfate fractionation followed by chromatography on hydroxylapatite, blue-sepharose, and SV40 DNA-sepharose. The SV40 DNA-sepharose was optimized for the binding of T-antigen by the covalent attachment of the SV40 DNA at its BamHI site to cyanogen bromide activated sepharose. The most highly purified T-antigen appeared as a single polypeptide of 94 K daltons by polyacrylamide gel electrophoresis.     

Evidence for Simian Virus 40 (SV40) Coding of SV40 T-Antigen and the SV40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus Type 2-SV40 Hybrid Viruses

HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND1, Ad2(+)ND2, Ad2(+)ND4, and Ad2(+)ND5) synthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methionine-containing tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific protein (100,000 daltons) from Ad2(+)ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2(+)ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virus-infected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and -transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.     

SV40 T-antigen is a histocompatibility antigen of SV40-transgenic mice

Although the extensive family of non-H-2 histocompatibility (H) antigens provides a formidable barrier to transplantation, the origin of their encoding genes are unknown. Recent studies have demonstrated both the linkage between H genes and retroviral sequences and the ability of integrated Moloney-murine leukemia virus to encode what is operationally defined as a non-H-2 H antigen. The experiments described in this communication reveal that skin grafts from an SV40 T-antigen transgenic C57BL/6 mouse strain are rejected by coisogenic C57BL/6 recipients with a median survival time of 49 days, which is comparable to those of many previously defined non-H-2 H antigens. The specificity of this response for SV40 T-antigen was demonstrated by the identification of SV40 T-antigen-specific cytolytic T lymphocytes and antibodies in multiply-grafted recipients. Although these cytolytic T lymphocytes could detect SV40 T-antigen on syngeneic SV40-transformed fibroblasts, they neither could be stimulated by splenic lymphocytes from T-antigen transgenics nor could they lyse lymphoblast targets from T-antigen transgenics. These observations suggest a limited tissue distribution of SV40 T-antigen in these transgenics. These results confirm the role of viral genes in the determination of non-H-2 histocompatibility antigenes by the strict criteria that such antigenes stimulate (1) tissue graft rejection and (2) generation of cytolytic T lymphocytes. Furthermore, they suggest that the SV40 enhancer and promoter region can target expression of SV-40 T-antigen to skin cells of transgenic animals.     

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.     

SV40-immortalized human fibroblasts as a source of SV40 infectious virions

Human fibroblasts immortalized by Simian Virus 40 (SV40) are widely employed for cell and molecular biology model of study. Indeed, SV40 transmission to humans was believed to occur only under exceptional situations. The oncogenic potential of SV40 in laboratory animals is well established, whereas its involvement in human carcinogenesis is still a matter of active investigations. A recent report links SV40 exposure with the development of a brain tumor in a laboratory researcher. In previous studies, episomal viral DNA was detected in SV40 stably transformed and immortalized fibroblast cell lines. In this study, we report molecular and biological characterizations of SV40 DNA in human fibroblast cells. Our results indicate that SV40 is able to establish a persistent infection in long-term immortalized human fibroblasts, resulting in the production of an infectious viral progeny, which is able to infect both monkey and human cells. These data indicate that SV40-immortalized human fibroblasts may represent a source of SV40 infection. To avoid the SV40 infection, careful attention should be given by operators to this SV40-cell model of study.     

Binding studies of SV40 T-antigen to SV40 binding site II.

SV40 T-Antigen binding site II was synthesized, cloned and analyzed for its ability to bind purified SV40 T-antigen. We report the binding constant of T-antigen for isolated site II. Using a filter binding assay the calculated binding constant was 6-8 fold less efficient than site I previously reported. Binding constants were calculated using two methods. The first was a direct calculation using a protein titration curve (KD). The second was by the ratio of measured association and dissociation rates. Both methods gave similar constants. Protection studies with SV40 T-antigen on the T-antigen binding sites in the wild-type array demonstrated that the binding constants of site I and site II are similar to those calculated for the individual sites. These results demonstrate that SV40 T-antigen does not bind cooperatively to sites one and two as earlier believed and are in agreement with recent observations emanating from several laboratories.     

Random integration of SV40 in SV40-transformed, immortalized human fibroblasts

We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.     

SV40-transformed simian cells support the replication of early SV40 mutants

CV-1, an established line of simian cells permissive for lytic growth of SV40, were transformed by an origin-defective mutant of SV40 which codes for wild-type T antigen. Three transformed lines (COS-1, -3, -7) were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replication of pure populations of SV40 mutants with deletions in the early region. One of the lines (COS-1) contains a single integrated copy of the complete early region of SV40 DNA. These cells are possible hosts for the propagation of pure populations of recombinant SV40 viruses.     

Temperature sensitive cells in the study of SV40 lysis versus SV40 transformation

Temperature sensitive variant clones of African green monkey kidney cell line (BSC-1) have been isolated which were transformed at a high frequency by SV40 at the restricted temperature, but were lytic to SV40 infection at the permissive temperature. Loss of contact inhibition and cell proliferation at the restricted temperature appeared to be in some way related to the synthesis of T antigen in these variant cell lines.     

Co-transfected SV40 origin of replication activates expression from SV40 promoterless constructs.

Co-transfection with expression plasmids is widely used to control DNA uptake efficiency in transient transfection experiments. However, a number of problems have been associated with their use. Here, we describe the activation of expression of constructs not containing the simian virus 40 (SV40) origin of replication (ori) by co-transfection in COS-7 cells with plasmids containing the SV40 ori. This effect has consequences for the use of such plasmids to control transfection efficiency.     

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.     

Specificities of killing by T lymphocytes generated against syngeneic SV40 transformants: studies employing recombinants within the H-2 complex.

T lymphocyte effectors to syngeneic SV40-transformed cells, generated by secondary in vitro sensitization of immune spleen cells, lyse SV40 transformed targets that are syngeneic at the H-2 locus. In this study we have employed recombinants within the H-2 region to examine in detail this H-2 specificity. H-2b effectors were found to lyse SV40-transformed targets from recombinants bearing either H-2Kb or H-2Db.H-2k effectors recognized only SV40-transformed H-2Kk, and not H-2Dk target cells. By using the same protocol for sensitization, no effector cells could be detected in H-2d mice. Effectors generated in H-2 recombinant mice showed that the response capacity resides with K and D. For example, HTG, which is H-2d except at the D locus (H-2Db), produced effector cells specific for SV40-transformed H-2Db targets. Thus, the secondary in vitro response to SV40 transformants was found to depend only on the K and D alleles and not to be modified by the I region to any measurable extent.     

Comparison of spontaneous and ultraviolet-induced mutagenesis on naked SV40 DNA and SV40 virus

Molecular aspects of mutagenesis in mammalian cells have been essentially analyzed using biological probes such as viruses and shuttle vector. Although the main data concerning the specificity of carcinogen-induced mutations are similar, the observed spontaneous mutation frequencies are significantly different when using one or the other model. This frequency is considerably higher with shuttle vectors than with viruses. We have performed an analysis of mutagenesis in order to determine if the obligatory transfection step associated with shuttle vector technology was responsible for the high mutation frequency found with these molecules. For this purpose simian virus 40 (SV40) genome used as virus or as naked DNA was introduced into permissive cells by viral infection or DNA transfection respectively. Our results show that transfection alone does not induce a higher mutation frequency on SV40 DNA the virus infection. Moreover, we have shown that the ultraviolet-light induced mutation spectrum was similar on the SV40 VP1 gene after viral infection or DNA transfection.