cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication
Journal of Virology
Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of
... d replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA. Mammalian cells transformed by simian virus 40 (SV40) harbor viral DNA sequences integrated in their chromosomal DNA and, in general, constitutively express SV40 large T antigen (for a review, see reference 48). T antigen is able to induce and maintain cell transformation, in part through its association with cellular proteins such as p53 and the retinoblastoma susceptibility gene product RblO5 (7; for a review, see references 24 and 48), although our understanding of the transformation process remains very incomplete. Clearly, however, activities of T antigen required for productive infection of monkey cells, such as initiation of viral DNA replication, are not essential for initiation or maintenance of cell transformation (48). The fact that many SV40-transformed cells express T antigens defective in their ability to initiate viral DNA replication has led to speculation that cell transformation by SV40 selects for expression of T antigens that maintain transformation but are no longer able to replicate SV40 DNA, perhaps thereby avoiding rearrangements of chromosomal DNA induced by T antigen (48). Alternatively, because T-antigen replication functions are not required to initiate or maintain cell transformation, replication-defective mutations in T antigen can accumulate in transformed cells (48) . Upon fusion of many SV40-transformed cell lines with permissive monkey cells, viral origins are activated by the resident T antigen, followed by amplification and recombination to yield free rescued SV40 DNA (for a review, see reference 48). This mobilization depends on the presence of an intact origin region and a replication-competent T antigen in the transformed cells (20, 41). In the event that the resident T antigen is replication defective, SV40 DNA can be rescued by fusion with COS1 monkey cells, which constitutively express a replication-proficient T antigen (20, 21). A number of replication-defective T-antigen mutants isolated by this method have proved useful in elucidating the biochemical activities of T antigen required for viral DNA replication, such as SV40 DNA binding and ATPase-helicase activities (for a review, see references 8 and 55). The SV40-transformed mouse line VLM (66) expresses large T and super T antigens able to bind specifically to both major sites in the SV40 control region and to cleave ATP (33). Fusion of VLM cells with TC7 cells yielded little free SV40 DNA, whereas viral DNA was efficiently rescued from VLM-COS1 fusions, suggesting that the VLM T antigens were almost inactive in replication (33). Thus, it seemed likely that identification of the presumed mutation in VLM T antigen could provide genetic evidence for other biochemical activities possibly involved in SV40 replication. We report here the characterization of a prototype clone rescued from VLM-COS1 fusions and compare it with genomic VLM DNA. Contrary to prediction, no defect in the origin function, early gene expression, or replication proficiency of the rescued T-antigen gene could be found. However, we demonstrate that mouse DNA sequences present in the SV40 late region of the rescued clone and derived from sequences flanking the SV40 integration site in VLM DNA suppress replication of SV40 plasmid DNA in uninfected monkey cells in an orientation-dependent manner. Furthermore, integrated viral DNA carrying the suppressor sequences in cis was rescued fivefold less efficiently from newly created transformed rat cell lines than viral DNA lacking the suppressor elements. MATERIALS AND METHODS Cells. All cells were cultured in Dulbecco modified Eagle medium (DMEM) (GIBCO-Bethesda Research Laboratories, Inc., Eggenstein-Leopoldshafen, Federal Republic of Germany) supplemented with 5% fetal calf serum (Hyclone; Greiner, Nurtingen, Federal Republic of Germany). VLM (67), TC7, a subline of CV1 African green monkey kidney cells (49), Rat2 (60), and COS1 (20) cells were described. VLM cells were cloned in soft agar prior to construction of genomic libraries. Seven of eight cell clones analyzed by genomic blotting with SV40 DNA probes revealed the same viral DNA integration pattern described previously (33) (data not shown).