Enhancement of Transcription of the SV40 Genome in Mouse Embryo Cells by Pretreatment with 5-Iodo-2'-deoxyuridine

Abstract

SUMMARYTreatment of mouse embryo (ME) cells with 5-iodo-2'-deoxyuridine (IdUrd) before infection with SV40 virus, enhances T-antigen (T-Ag) production as detected by immunofluorescence and complement fixation. Cellular DNA and RNA synthesis are inhibited in both SV40 and mock-infected cells after IdUrd treatment. The analogue pretreatment significantly increases the amount of radiolabelled nuclear and cytoplasmic SV40-specific RNA and the RNA polymerase activity of the viral transcriptional complexes of the Sarkosyl supernatants, suggesting that the enhancement of SV40 T-Ag production in infected pretreated ME cells results from an increased synthesis of early virus RNA.It has been reported previously (Su~trez et al., 1976(Su~trez et al., , 1977) that treatment of fully, as well as semi-permissive, cells with 5-iodo-2'-deoxyuridine (IdUrd) before treatment with SV40 virus or virus DNA, resulted in a marked increase of T-antigen (T-Ag) production and virus yield. No effect of the drug on the replication of the virus was found when the experiments were carried out with non-permissive (mouse or Syrian hamster embryo) cells (Suarez et al., 1976). Experiments performed to determine whether IdUrd affects adsorption, penetration or decapsidation of SV40 in permissive or semi-permissive cells indicated that the analogue acts between the arrival of the virus DNA in the nucleus and T-Ag synthesis. Moreover, the enhanced production of T-Ag persisted in permissive cells even if cytosine arabinoside (Ara-C) was immediately added after infection (Su/lrez et al., 1977). These results suggest that the halogenated pyrimidine acts at an early step of the SV40 cycle, perhaps at the level of early virus transcription or translation.Mouse primary or secondary cultures are non-permissive for SV40. Infection with the virus does not lead to synthesis of virus DNA or production of late virus polypeptides, but substantial amounts of SV40 T-Ag and the early SV40 RNA which encodes this protein(s) can usually be detected. This infection leads, within a few weeks, to the appearance of SV40-transformed cell colonies (Smith et aL, 1979). Mouse cells infected with SV40 are therefore particularly useful for investigating the effects of IdUrd pretreatment on the expression of SV40 early functions.Mouse embryo (ME) fibroblasts were prepared from 12-to 14-day-old embryos of Swiss albino mice. Cultures were routinely grown in Eagle's minimal essential medium (MEM, Eurobio, Paris, France) supplemented with 10% calf serum. Secondary ME cells suspended in growth medium supplemented with 10% calf serum, were seeded at 4 × 106 or 2 x l0 s cells/culture in 10 cm diam. plastic dishes, in the presence or absence of 100/zg/ml IdUrd respectively, and incubated at 37 °C. Four days later, the medium was removed from the cultures and after trypsinization, the number of cells was determined. It was virtually the same in treated and untreated cultures. The cells were then infected with SV40 virus at 50 p.f.u./cell. After 2 h adsorption at 37 °C, excess virus inoc...