ABSTRACT
Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or Clara cells caused by a type D/B chimeric retrovirus, jaagsiekte sheep retrovirus (JSRV). Here we report the isolation, sequencing, pathogenicity, and integration site of a JSRV provirus isolated from a sheep lung tumor cell line (JS7). The sequence of the virus was 93 to 99% identical to other JSRV isolates and contained all of the expected open reading frames. To produce virions and test its infectivity, the JS7 provirus (JSRV(JS7)) was cloned into a plasmid containing a cytomegalovirus promoter and transfected into 293T cells. After intratracheal inoculation with virions from concentrated supernatant fluid, JSRV-associated OPC lesions were found in one of four lambs, confirming that JSRV(JS7) is pathogenic. In JS7-cell DNA, the viral genome was inserted in the protein-coding region for the surfactant protein A (SP-A) gene, which is highly expressed in ATII cells, in an orientation opposite to the direction of transcription of the SP-A gene. No significant transcription was detected from either the viral or the SP-A gene promoter in the JS7 cell line at passage level 170. The oncogenic significance of the JSRV proviral insertion involving the SP-A locus in the JS7 tumor cell line is unknown.
Subject(s)
Jaagsiekte sheep retrovirus/genetics , Proteolipids/genetics , Proviruses/genetics , Pulmonary Adenomatosis, Ovine/virology , Pulmonary Surfactants/genetics , Virus Integration/genetics , Animals , Base Sequence , Binding Sites , Cell Line, Transformed , DNA, Viral , Humans , Jaagsiekte sheep retrovirus/isolation & purification , Jaagsiekte sheep retrovirus/pathogenicity , Lung Neoplasms , Molecular Sequence Data , Proviruses/isolation & purification , Pulmonary Surfactant-Associated Protein A , Pulmonary Surfactant-Associated Proteins , Sheep , Tumor Cells, CulturedABSTRACT
An efficient and reproducible technique is described for the isolation of transformed sheep pulmonary adenomatosis cells. It includes three basic steps: prolonged trypsinisation to kill fibroblasts, magnetic removal of macrophages and adherence to remove the rapidly adherent cells. The resultant preparations of lung cells were enriched to 96.6 per cent type 2 pneumocytes.