Cloning and Sequencing of Viral Integration Site in HumanFibroblasts Immortalized by Simian Virus 40

Wehave analyzed cellular DNAsequences at the viral genome integration site in a human fibroblast cell line VA13immortalized by simian virus 40 (SV40). The computer analysis of the junctional cellular DNAsequences did not show any homology to the DNAsequences previously reported. This suggests that immortalization by SV40was not induced by the destruction of any knownoncogene or antioncogene at the integration site. Wedid not find the precise substantial sequence homology at the junctional site between the cellular DNAand SV40 DNA, indicating that the recombination mechanism involved does not require precise sequence homology and therefore, SV40 genome was probably not integrated by homologous recombination. Short direct and inverted repeats of 5 to 29 nucleotides were found in the junctional cellular and SV40 DNA. Cellular DNAabutting SV40 DNAwas found by the Northern blot analysis to be expressed in diploid human fibroblasts and SV40-transformed cells. The nature of this RNAis now under study. Unlike rodent cells which easily form continuously growingcultures either spontaneously or after viral infection (45), diploid human fibroblasts have a limited lifespan in vitro (20, 21) and are resistant to transformation to an immortal form not only by spontaneous change but also physical or chemical means (27, 37). When infected with simian virus 40 (SV40), human fibroblasts can easily be transformed and can grow longer than their normal counterpart but eventually deteriorate and perish (13, 14, 24). In all SV40-transformed cells, at least a part of the viral DNA is persistently integrated in host cellular DNA (46) and the functional large T antigen is expressed until the very end of their lifespan (14). In very rare cases, however, an immortalized cell arises from these SV40-transformed cells (13, 14, 24). Taken together, the above suggests that large T antigen is required to transform humanfibroblasts but that it is not enough to immortalize them. Someauthors have suggested that the integration of the SV40 DNAin SV40-transformed and immortalized human fibroblasts occurs at random locations. However, these authors investigated integration sites only by Abbreviations: SV40, simian virus 40; MCF-PBS,magnesium, calcium free phosphate-buffered saline; TE, 10 mMTris-HCl and 1 mM EDTA, pH 8.0 analyzing the assignment of SV40 DNAto specific chromosomes (7, 8, 31) or by analyzing Southern blotting patterns (5, 18, 25). Their arguments can be criticized on several points. First, if short stretches of specific cellular DNA sequences would border viral insertions, they could not be detected by Southern blot analysis. Moreover, the different alterations of cellular DNA after the integration of a virus genome at the specific sites should produce restriction fragments of various sizes in different cell lines. Furthermore, when differently rearranged SV40 genomes are integrated at many sites including the same hot spot, the Southern blotting pattern would not be same. Although in the case of rat cells, it has been reported that SV40 DNAis not integrated at the specific site by sequencing the junctional nucleotides sequences of only 20 bases or so (15, 44), no one has reported the DNAstructure of SV40DNAintegration site at the nucleotide level in immortalized humanfibroblasts. In our present study, we cloned the DNAfragments containing the integrated viral DNAfrom SV40-transformed human fibroblast VA13 clone 1 cells into procaryotic vectors and determined the DNAsequences encompassing the junctions between SV40 and cellular DNA.Also, we performed the Northern blot analysis (1, 32) to investigate the expression of the junctional cellular DNAsequence and found they were expressed in