Tumorigenic transformation induced by a specific fragment of DNA from herpes simplex virus type 2.

Abstract

Inactivated herpes simplex virus type 2 (HSV-2) (1, 2) and low concentrations of native, noninactivated viral DNA (3, 4) induce the neoplastic transformation of mammalian cells in culture. Transformation appears to be mediated by fragment(s) of the HSV-2 genome: (i) mechanically sheared HSV-2 DNA (Mr 9 X 106) induces neoplastic transformation at a frequency identical to that observed with native DNA (4), (ii) hamster cells transformed by UV-irradiated HSV-2 retain a fraction (8-32%) of the viral DNA sequences (5), and (iii) virus transforming activity is more resistant than its infectivity to radiation damage (1, 2). However, the oncogenic fragment in the HSV-2 genome has not yet been identified.Provided that oncogenic activity does not depend on distant noncontiguous genes and that suitable restriction endonucleases can yield appropriate fragments of HSV-2 DNA that do not express lytic functions, it seems reasonable to propose that neoplastic transformation should be demonstrable with a restriction endonuclease fragment. The data described in this report support the validity of this proposal.MATERIALS AND METHODS Cells and Virus. Syrian hamster embryo (SHE) cells were grown in ERM medium with 10% fetal bovine serum (3). Vero and HEp-2 cells were grown in medium 199 with 10% fetal bovine serum. HSV-2 strain S-1, a human cervical tumor isolate (6), was plaque-purified and grown in HEp-2 cells at a low (0.1 plaque-forming unit/cell) multiplicity of infection. HSV-2 strain 333 was obtained from R. Duff.DNA Purification. Viral DNA was purified from Vero cells infected with HSV-2 strains S-1 or 333 by buoyant density centrifugation in NaI gradients containing ethidium bromide as described (3, 7). To isolate fragments, we crushed gel slices in buffer (10 mM Tris-HCl/1 mM EDTA/0.1% NaDodSO4, pH 8.0) and incubated them overnight at 45°C. Agarose was removed by centrifugation at 10,000 rpm for 20 min, and the supernatant was extracted with phenol/chloroform/isoamyl alcohol and dialyzed against Hepes-buffered saline, pH 7.05 (9). DNA concentration was determined by UV absorbance at 260 nm. Recovery was approximately 75%.

Identification of a Herpes Simplex Virus Type 1 Polypeptide Which Is a Component of the Virus-induced Ribonucleotide Reductase

“…Two regions of the HSV-2 genome which morphologically transform cells in vitro have been identified (Reyes et al, 1979;Jariwalla et al, 1980;Galloway & McDougall, 1981). One region v.G.…”

Immortalization and neoplastic transformation of normal diploid cells by defined cloned DNA fragments of herpes simplex virus type 2.

“…Transfection with isolated restriction-enzyme fragments of viral DNA has identified one set of transforming sequences in HSV-1 DNA (7). On the other hand, two distinct transforming regions were identified in HSV-2 DNA (8)(9)(10). One of these, the Bgl II fragment C, was found to transform normal diploid Syrian hamster embryo (SHE) cells in the continuous passage assay (8).…”

Localization of the Coding Region for a 35000 Dalton Polypeptide on the Genome of Herpes Simplex Virus Type 2

“…In a number of laboratories, the presence of HSV-specific antigens has been detected in transformed cells, including membrane glycoproteins and the non-structural proteins VPI43 and ICP10 (Camacho & Spear, 1978;Duff& Rapp, 1973;Lewis et al, 1982;Reed et al, 1975;Flannery et al, 1977;Jariwalla et al, 1980). We have recently demonstrated that nine polypeptides with molecular masses of 110, 84, 77, 47, 41.5, 38, 37.5, 35 and 32 kilodaltons are precipitated from transformed or tumour-induced hamster cells by anti-HSV serum but not by control serum (Suh et al, 1980).…”

“…Using focus formation assays, the BgllI N restriction fragment (0.582 to 0-628 map units) has been shown to tra:asform BALB/3T3 (Reyes et al, 1979), primary rat, and NIH/3T3 (Galloway & McDougall, 1981) cells. Jariwalla et al (1980) selected transformed cells which grew out of continuous passage after exposure to fragments of HSV-2 DNA. These experiments identified a fragment with transforming activity mapping between map coordinates 0-43 and 0.58.…”

“…Inactivated herpes simplex virus type 2 (HSV-2) (Duff & Rapp, 1971) and DNA or cloned DNA fragments from HSV-2 (Wilkie et al, 1974;Galloway & McDougall, 1981 ;Jariwalla et al, 1980;Macnab, 1974) have been used for oncogenic transformation of rodent fibroblasts in culture. In a number of laboratories, the presence of HSV-specific antigens has been detected in transformed cells, including membrane glycoproteins and the non-structural proteins VPI43 and ICP10 (Camacho & Spear, 1978;Duff& Rapp, 1973;Lewis et al, 1982;Reed et al, 1975;Flannery et al, 1977;Jariwalla et al, 1980).…”