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The viral DNA sequences in mouse C127 cells transformed by bovine papillomavirus type 1 (BPV-1) virions, by full-length linear BPV-1 DNA, or by a defined transforming subgenomic DNA segment of BPV-1 were examined by reassociation kinetics and blot hybridization. In all cases, the transformed cells contained multiple copies of BPV-1 DNA, present exclusively as supercoiled or nicked circular extrachromosomal molecules or as a slowly migrating complex of circular viral DNA molecules. In the transformed cell lines established from cells transfected with full-length linear BPV-1 DNA, there was recircularization of the input DNA which in some cases resulted in the loss of the restriction site used in the linearization of the DNA. In the transformed cell lines established with the defined subgenomic segment there was circularization of the DNA accompanied by the acquisition of new sequences or duplication and rearrangement of the BPV-1 sequences. In contrast to other well-studied virus transformation systems, no integration of the BPV-1 genome into the host chromosome could be detected under conditions sensitive enough to detect 0.1-0.2 viral genome equivalent. It was concluded that maintenance of transformation may be mediated by nonintegrated viral DNA.

We have studied tumorigenic transformation of mouse tissue culture cells by bovine papillomavirus (BPV) as a model of papillomavirus-induced cell proliferation. When BPV or its 7.9-kb full-length viral DNA genome induces focal transformation of mouse calls, the viral DNA is maintained in the transformed cells as multiple extrachromosomal copies. The transforming capacity was initially localized to a 69% subgenomic fragment of the viral DNA genome. We have further characterized the BPV DNA sequences that can encode the transforming function by generating and analyzing the transforming activity of a series of BPV DNA deletion mutants. The results indicated that two discontinuous segments of the viral DNA are required for transformation. One segment, near the 5' end of the 69% transforming fragment, probably represents a control element of the viral DNA. The second segment, which lies within the 3' end of the 69% fragment, encodes transforming sequences of the viral DNA. A retroviral control element (the long terminal repeat DNA. A retroviral control element (the long terminal repeat DNA of Harvey murine sarcoma virus) will activate the 2.3-kb segment at the 3' end of the 69% fragment to transform the mouse cells.

The effect of mouse L-cell interferon on bovine papillomavirus type 1 (BPV-1) transformation of murine cells was examined. Mouse interferon reduced the level of BPV-1-induced transformation of mouse C127 cells by 95%. Long-term treatment of established BPV-1-transformed mouse cell clones with mouse L-cell interferon led to a decrease in the average number of the plasmid viral genomes present in these cells to 1/3 to 1/8. Although revertant lines could not be isolated from these lines in the absence of treatment with interferon, flat revertants were easily selected from two independent clonal transformed lines carried for 60 generations in the continued presence of 200 units of interferon per ml. These flat revertants had the biological characteristics of nontransformed C127 cells and could be retransformed by BPV-1. Southern blot hybridization failed to detect BPV-1 DNA in any of eight independent revertant lines examined under conditions that could detect 0.2 copies per cell. We conclude that interferon treatment has resulted in a selective reduction of the amount of extrachromosomal BPV-1 DNA in transformed cells and has cured some treated cells completely of their viral DNA.

The papillomaviruses, which are widely distributed in nature, typically induce benign skin tumours (warts) in their natural hosts (reviewed in ref. 1). These viruses are members of the papovavirus group, as are the polyomaviruses2. In contrast to polyomaviruses such as SV40, polyoma and BK (reviewed in ref. 3), the papillomaviruses have not been extensively studied, primarily because of the lack of a suitable tissue culture system. In general, the proliferative skin changes induced by the papillomaviruses are limited to the epidermal cells, and the viruses have a very narrow host range. However, some bovine papilloma viruses (BPV) are exceptional in that they induce fibropapillomas in their natural host (cows); these BPV can also induce cellular transformation in tissue culture4–6 and fibroblastic tumours in heterologous animals, including hamsters7, mice5 and horses8,9. BPV DNA from virions can also transform rodent cells and is tumorigenic in hamsters10. Recently, a tissue culture focus assay11 in mouse cells (NIH 3T3 and C127) has been described for those BPV which are the aetiological agents of fibropapillomas in cows. The virally transformed cells, which did not contain infectious virus, grew in soft agar and were tumorigenic in nude (nu/nu) mice. We12 have recently molecularly cloned the entire genomes of BPV-1 and BPV-2 (ref. 13) in Escherichia coli K12 using the certified plasmid vector pBR322 (ref. 14). We now demonstrate that these cloned DNAs can transform NIH 3T3 and C127 cells. Further, a molecularly cloned fragment which contains 69% of the BPV-1 genome can also induce cellular transformation.