Expression of integrated Rous sarcoma viruses: DNA rearrangements 5′ to the provirus are common in transformed rat cells but not seen in infected but untransformed cells.

Abstract

The study of Rous sarcoma virus (RSV)‐infected rat cell clones offers a novel approach to unravelling the mechanisms controlling eukaryotic gene expression. RSV‐transformed rat cell clones frequently contain duplicated proviral sequences immediately upstream of an intact provirus. This category of proviral rearrangement is not seen in cells that remain untransformed after RSV infection nor in subsequently segregating transformants. These results suggest that such rearrangements occur during or soon after proviral integration, and that they may favour early proviral expression.

On the structure, genesis and significance of DNA duplications at the Rous sarcoma proviral insertion sites in Rat-1 cells

“…To summarize, in this and previous work (Gillespie et al, 1985;Green et al, 1986;Fincham & Wyke, 1991) we have analysed a total of 47 different Rat-1 lines containing single transcribed proviruses and in 32 of these there are DNA duplications invariably 5' to the provirus. Falcone et al (1992) have recently observed comparable duplications, as well as proviral amplification, in BALB/c mouse cells transformed by RSV.…”

“…The vast majority (99.9~0 or more) are simple insertions of single proviruses at chromosomal sites that are not distinguishable from random, and these proviruses are not detectably transcribed (Green et al, 1986;Fincham & Wyke, 1991). About 30~ of the small remainder are also simple single insertions, but they are expressed.…”

“…Their insertion sites are clearly non-random (Fincham & Wyke, 1991), almost all being closely 3' to sites for BssHII, SaclI or NarI that are predominantly found in cellular CG-rich islands (Lindsay & Bird, 1987). However, most expressed proviruses are not simple insertions, displaying instead duplications of proviral, and occasionally host, DNA to the 5' side of, and contiguous with, a complete proviral unit (Gillespie et al, 1985;Green et al, 1986). The biased location of these rearrangements and their absence at non-transcribed proviral loci suggested that they may encourage proviral transcription by attenuating the influence of the insertion site.…”

“…Several transcribed proviruses with contiguous 5' duplications have been identified previously (Gillespie et al, 1985;Green et al, 1986); this collection was enlarged by newly infecting Rat-1 cells with B77 strain RSV and isolating foci whose transformed morphology indicated proviral transcription. The number and structure of proviruses in these cells were characterized by digesting genomic DNA with a variety of restriction enzymes and hybridizing digests with the subgenomic proviral probes shown in Fig.…”

CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression

“…Fewer than 0.1% of RSV provirus-containing mammalian cells displays morphological transformation by virtue of the v-src oncogene (6,7). The vast majority of RSV-infected cells harbors transcriptionally silent proviruses with undetectable amount of viral RNA (7,8). Even in transformed mammalian cells, the proviral expression often tends to spontaneous silencing and segregation of revertant cell clones with nontransformed phenotypes (9)(10)(11).…”