Characterization of Transforming Viruses Rescued from a Hamster Tumour Cell Line Harbouring the v-src Gene Flanked by Long Terminal Repeats

Abstract

SUMMARY"The organization of proviruses derived from infecting transforming viruses rescued from hamster tumour cells was studied. Southern blot analysis indicated that the provirus from the F6 cell line was organized as long terminal repeat (LTR)-src-LTR, and S1 mapping experiments suggested that it was probably derived by reverse transcription.ofsrc mRNA followed by integration. In the E6 cell line, the provirus unit was arranged as LTR-A gag-src-LTR, indicating a recombination event between the rescued transforming virus and the helper virus. These results suggest that transforming defective viruses containing only the src gene can be rescued from nonpermissive mammalian cells.

The LTR, v-src, LTR provirus generated in the mammalian genome by src mRNA reverse transcription and integration

“…Transforming virus was detected 12 days after transfection (7 x 101 focus-forming units/ml) and reached a titer of 6.5 x 102 focus-forming units/ml after 4 weeks of culture. The relatively low titer of this transforming virus has already been noticed in virus rescue experiments (8). This indicates that in spite of the presence of the main structures required for packaging in src mRNA (see below), some additional cis-acting and packaging signals of the gag region are absent (1, 2, 14, 27, 33).…”

“…It was demonstrated that this cryptic provirus is functional, because if properly complemented (by fusion with chicken fibroblasts preinfected with Rous-associated virus 1), transforming viruses can be rescued from H-19 cells which encapsidate RNA having the character of nonpolyadenylated src mRNA (37). According to restriction enzyme analysis and Si mapping, the proviral structures of most of the rescued viruses, as well as of the original H-19 provirus, corresponded to that of reverse-transcribed src mRNA (8,37). The aim of this work was to elucidate the structure of the LTR, v-src, LTR provirus integrated in the hamster genome to understand its genesis.…”

A transformation-competent recombinant between v-src and Rous-associated virus RAV-1

“…We have previously described a functional proviral structure that consists of LTR, v-src, LTR sequences integrated in the hamster tumor H-19 and that, according to restriction mapping and sequencing, corresponds to the reverse-transcribed src mRNA (4,10,30). This provirus can be rescued if the nonpermissive H-19 cells are complemented by fusion with Rous-associated virus RAV-1-infected chicken fibroblasts and the v-src proviral transcript is encapsidated in RAV-1 virions (10, 27).…”

“…2). The 3.7-kb species hybridizes with the gag 2, src 11, and LTR 2 probes (10,15,19), detecting the nucleotide regions 532 to 1916, 8098 to 8671, and 8992 to 9335, respectively, of Rous sarcoma virus genomic structures (22), and represents the transcript of the whole E6 genome. The smallest species corresponds to the 2.7-kb src mRNA and hybridizes only to the long terminal repeat (LTR) and src probes.…”

“…RNAs were transferred on a Nytrone membrane (Schleicher & Schuell, Inc.) in 1x SSC (150 mM NaCl-15 mM sodium citrate, pH 7.0), and the membrane was then UV irradiated for 2 min with a Philips TUV 15-W tube at a 12-cm distance. For hybridization, 32P-labeled plasmid probes, gag 2, src 11, and LTR 2 (10,15,19), detecting specifically the viral genome regions spanning nucleotides 532 to 1916, 8098 to 8671, and 8992 to 9335, respectively, were used. arrangement of cellular DNA direct repeats in the ancestral H-19 provirus (4).…”