Isolation of Defective Lysogens from Simian Virus 40-transformed Mouse Kidney Cultures

Abstract

Rescue of simian virus 40 (SV40) from hamster and murine cell lines transformed by nonirradiated or by ultraviolet (UV)-irradiated SV40 (10-s to 10-s survival) was studied. A combination of tests was employed to detect induction of SV40 synthesis: (i) co-cultivation with susceptible monkey kidney (CV-1) cells; (ii) treating mixtures of transformed and CV-1 cells with UV-irradiated Sendai virus (UV-Sendai) prior to co-cultivation; and (iii) plating untreated or UV-Sendaitreated mixtures of transformed and CV-1 cells with freshly trypsinized CV-1 cells. The first and second tests provided a measure of the total infectious SV40 yield per culture, and the third test provided a measure of the frequency of induction (fraction of transformed cells giving rise to infectious centers). With the combination of tests, SV40 was rescued in all trials from TSV-5 hamster cells, mKS-BU100 mouse cells, and from several lines of mouse kidney cells transformed by UV-irradiated SV40 (mKS-U lines). The frequency of induction was about 7 X 10-2 for TSV-5 cells, about 3 X 10-3 for mKS-BU100 cells, greater than 10-4 for the mKS-U lines which were "good" yielders, and about 10-s to 10-4 for the mKS-U lines which were "average" yielders. SV40 of a plaque type different from parental virus was rescued from four of the mKS-U cell lines. Virus was also easily rescued from: (i) tumor cells produced from the mKS-A line of transformed mouse kidney cells; (ii) mouse kidney cells transformed by SV40 which had been rescued from mKS-BU100 cells; and (iii) tumor cells (HATS) which had been produced by inoculating newborn hamsters with SV40 rescued from mKS-BU100 cells. The frequency of induction of HATS cells was of the same order of magnitude as the frequency of induction of TSV-5 cells. In a study of the kinetics of virus induction, it was shown that SV40 could be detected 28, 40, and 48.5 hr after UV-Sendai treatment of mixtures of CV-1 and TSV-5, HATS, or mKS-BU100 cells, respectively. Although all of the mKS-U lines contained the SV40-specific tumor antigen, some were poor virus yielders (SV40 was recovered in less than 50% of the trials) and five lines were rare virus yielders (SV40 recovered only once in four or more trials). Forty-eight mKS-U lines were nonyielders; SV40 was never recovered by any test used thus far. UV-Sendai-treated mixtures of pairs of nonyielder mKS-U lines with CV-1 cells also did not yield infectious virus. Various factors affecting rescue have been discussed. The mKS-U lines which were poor virus yielders, rare yielders, or which never yielded virus have been classified tentatively as "defective lysogens" which contain mutational lesions at loci essential for detachment of SV40 from integration sites or for SV40 replication, or for both. A number of simian virus 40 (SV40)-transthesis, probably by increasing cell fusion and formed cell lines which do not spontaneously heterokaryon formation. Cloning experiments produce virus can be induced to do so when and characterization of SV40 rescued from these cocultivated with s...

Transplantable mouse tumor line induced by injection of SV40‐transformed mouse kidney cells

“…In contrast to other systems, the present system uses a readily transplantable mouse tumor, and, as hosts, young adult mice which need not be subjected to pretreatment with immunosuppressive agents or thymectomy. The system is currently being used to compare the capacities of wild-type SV40 and various rescued SV40 strains to induce rejection of mKS-A TU-5 cell transplants Dubbs and Kit, 1968). Mutant virus strains lacking the ability to immunize mice against the mKS-A TU-5 tumor cells are of obvious interest in relation to the biological function of the transplantation antigen and its possible role in oncogenesis.…”

“…SV40 clone 307L was propagated and assayed in monolayer cultures of CV-1 cells (Kit et al, 1966b). Sendai virus was grown in the allantoic cavity of 10-to 11-day-old embryonated eggs (Dubbs and Kit, 1968).…”

“…The mKS-US, mKS-U7, mKS-U18, mKS-U13 and mKS-U17 cell lines, were derived from primary mouse (non-inbred) kidney cultures transformed by ultraviolet (UV)-irradiated SV40 (Dubbs and Kit, 1968). The mKS-U13 line readily yields infectious virus after fusion with CV-1 cells in the presence of UV-irradiated Sendai virus.…”

“…Tumor cells cultured in vitro were mixed with CV-1 cells in the presence of UV-irradiated K I T ET AL. Sendai virus as previously described (Dubbs and Kit, 1968). Aliquots of the UV-irradiated Sendai treated cell mixtures were planted in 60 mm Petri dishes along with lo6 freshly trypsinized CV-I cells in 5 ml growth medium, and incubated overnight.…”

“…BALB/c mice were also twice immunized with lo6 monkey kidney (CV-1) cells. Finally, BALB/c mice were twice immunized with lo6 mKS-VS (non yielder), mKS-V7 (non yielder), or mKS-V18 (rare yielder) cells (Dubbs and Kit, 1968). The latter three transformed cell lines do not produce tumors in mice.…”