Herpes Simplex Virus Sequences Involved in the Initiation of Oncogenic Morphological Transformation of Rat Cells Are Not Required for Maintenance of the Transformed State

Abstract

SUMMARYWe have determined the herpes simplex virus (HSV) type 2 DNA sequences responsible for the initiation of morphological transformation and have investigated the retention and expression of these sequences in morphologically transformed cells and in tumours derived from these cells. All the transformed cells analysed were selected by a focus formation assay and are oncogenic in the inbred host rat. Cloned HindIII and BglII fragments from the HSV-2 genome were assayed for the ability to initiate morphological transformation of rat embryo cells. Only the HindIII a (map units 0.52 to 0-72) and the BgllI n (0'582 to 0.612) clones gave transformed foci. This shows that the BglII n region is responsible for initiation of transformation. Southern blot analysis of DNA extracted from these transformed cells and from turnouts derived from these transformed cells revealed that neither the BgllI n fragment nor fragments of 500 bp mapping within it are detected at the level of one copy per cell and therefore need not be retained in the cell to maintain the oncogenic phenotype. In addition there was no evidence of expression of the HSV-specified ribonucleotide reductase activity which is partially encoded within the BglII n fragment of HSV-2. We also analysed DNA from rat embryo cells transformed by ts mutants of HSV-2 (HG52) or HSV-1 (HFEM or 17) at non-permissive temperature or by virus at supraoptimal temperature or by sheared virus DNA and DNA from tumours derived from lines of these transformed cells. In addition, we cloned both transformed and tumour cell lines and analysed these similarly. In no case could we detect HSV DNA sequences at the level of one copy per cell.

Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus.

“…Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.It has been recognized since 1971 (12) Our interest in the induction of morphological transformation continues from our findings that transformed cells all react with antisera raised against HSV-infected cells (9,26,27 have shown that treatment of mouse embryo fibroblasts with the demethylating agent 5-azacytidine can lead to cell transformation.In this communication, we present evidence for the hypomethylation of DNA in a number of cell lines derived from foci morphologically transformed by HSV type 1 (HSV-1) or by the DNA from the transforming region of HSV-2 (the BglII N fragment). To demonstrate that this event is initiated by virus infection, we also report on the hypomethylation of host cell DNA synthesized during HSV-2 infection of primary rat embryo cells and provide evidence for the limited reincorporation of methylcytosine from degraded cellular DNA into viral DNA.…”

“…It has been recognized since 1971 (12) Our interest in the induction of morphological transformation continues from our findings that transformed cells all react with antisera raised against HSV-infected cells (9,26,27 have shown that treatment of mouse embryo fibroblasts with the demethylating agent 5-azacytidine can lead to cell transformation.…”

“…Rat embryo control cells were prepared from 18-day-old embryos of the Hooded Lister rat (9). Rat embryo Asyn+C cells are rat embryo cells transformed at nonpermissive temperature with the HSV-1 strain 17 temperature-sensitive, (ts) mutant tsA with morphology of syn+.…”

“…Our interest in the induction of morphological transformation continues from our findings that transformed cells all react with antisera raised against HSV-infected cells (9,26,27). The results demonstrate that infection with HSV induces the expression of immunologically competent cell polypeptides whose expression can also be detected in transformed cells.…”

“…These features include the structure of the transforming DNA involving the presence of insertion sequences (16) or altered repeat elements (22), biological carcinogenesis such as mutation or gene amplification (35,39), or even the involvement of the ribonucleotide reductase gene whose large and small subfragments map within the DNA sequences involved in the induction of morphological transformation (reviewed by Macnab [26]). What is reasonably clear is that viral DNA encoding a transforming protein is not retained in the cells (9,15).…”