Cell, Vol 38, 483-491, September 1984, CopyrIght 0 1984 by MIT 0092.8674/84/090483-09 $02.00/O Transformation Mediated by the SV40 T Antigens: Separation of the Overlapping SV40 Early Genes with a Retroviral Vector Michael Kriegler*, Carl F. Perez, Chris Hardy, and Michael Botchan Department of Molecular Biology University of California Berkeley, California 94720 Summary A murine retroviral vector has been used to separate physically the overlapping genes encoded by SV40. This minimal retroviral vector contains LTRs and other c&acting signals required for infectious RNA virus propagation. We placed the SV40 early region within this DNA and after transfection of cells pro- ducing helper Moloney murine leukemia virus, SV40 retroviruses (MV40) could be rescued. Cytoplasmic spliced large T and small t transcripts, as well as unspliced transcripts, are packaged into virions with equal efficiency. Pure SV40 large T retroviruses can be cloned from these heterogeneous virus stocks by secondary transformation of rodent cells. The large T retrovirus stocks morphologically transform primary or established mouse and rat lines with high efficiency. There is little difference in transformation either by agar assay or focus formation between retroviruses carrying both SV40 genes or large T alone. We present quantitative data that demon- strate that abortive transformation of rodent cells by SV40, transient expression of the transformed phe- notype after infection, is not manifested by MV40. Thus abortive transformation is not the result of a weakly dominant transforming gene, but rather of the normally inefficient mode of integration and early gene expression of SV40 upon infection of rodent cells. Introduction Differential splicrng of RNA derived from one transcription unit is a frequent expression strategy employed by animal viruses and some cellular genes. Overlapping genes serve to compact coding space by allowing common sequences to be mixed and matched in various ways to produce different protein products. The early region of SV40 pro- vides an example of overlapping genes. The 94 kd large T antigen and the 17 kd small t antigen share common amino termini but differ in their carboxy-terminal portion, a consequence of differential splicing of a single immature transcript (Berk and Sharp, 1978; Khoury et al., 1980). While only the large T antigen seems to be required in the viral lytic cycle, both of these proteins have been implicated In the transforming activity of SV40. The conventional approach to genetic separation of the activities of these peptides has involved the characterization of both deletion and point mutations (Tooze, 1980). * Current address Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Phlladelphla, PA 19111 A direct approach to the separation of overlapping genes is the synthesis and independent expression of cDNA copies of the spliced transcripts (Treisman et al., 1981; Rassoulzadegan et al., 1982, 1983). An alternative approach to the separation of the biochemical and biolog- ical activities of the products of overlapping genes could exploit the observation that recombinant retroviruses ex- cise the introns of genes inserted in vitro into their genomes (Shimotohno and Temin, 1982; Sorge and Hughes, 1982). One could insert a DNA fragment spanning a complex family of overlapping genes into a retroviral vector, trans- feet the appropriate helper virus producer cell, and, after packaging of the spliced transcripts, produce a population of infectious recombinant virions. Purification of a particular recombinant virus can proceed after infection of the ap- propriate indicator cell line to allow for either brochemical or biological screenrng of the infected cell population. We tested this methodology with the SV40 early region for two distinct reasons. First, stable transformation of rodent cells by SV40 is inefficient. Although abortive trans- formation can be detected in a high percentage of infected cells, only a minute fraction remains stably transformed (Stoker, 1968; Rubin et al., 1982; Benjamin et al., 1980). Furthermore, as a consequence of the host-cell specificity of the SV40 enhancer, it has been shown that after inte- gration in some chromosomal positions the SV40 early genes cannot be expressed at levels sufficient to manifest morphological transformation (Hanahan et al., 1980; Peru- cho and Wigler, 1980; Laimins et al., 1982; Kriegler and Botchan, 1983; Kriegler et al., 1983; Scholer and Gruss, 1984). After transient expression of the viral transforming genes, the viral DNA, if not integrated in an appropriate position, is either genotypically or phenotypically lost. Since the c-type retroviruses include high-efficiency stable inte- gration as a normal step in their replication cycle (Temin, 1980; Varmus, 1982) they are ideal vectors for high- efficiency transformation and thus provide a means for testing the models for SV40 abortive transformation. Our results show that SV40 contains dominant transforming genes capable of transforming every infected cell. Second, as the roles of large T and small t in morpho- logic transformation are not clear (Topp et al., 1980) in the event that the scheme outlined above for separation of alternatively spliced RNAs worked, we could then use direct biological selection for the SV40 transforming func- tion(s) without experimental bias. In this report, we present biochemrcal and brological data on two in vitro-generated SV40 retroviruses that encode large T and small t, and large T. The large T retrovirus represents an authentic early-region cDNA retrovirus and is capable of morpholog- really transforming rodent cells wrth high efficiency. Results Expression of the SV40 Early Region in Retroviral Vectors We designed a retroviral vector, pEVX, into which we could insert the SV40 early region. Our aim was to put the SV40