The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain.
Ovine pulmonary adenocarcinoma, caused by jaagsiekte sheep retrovirus (JSRV), is a naturally occurring retrovirus-induced pulmonary neoplasm of sheep. We report here that expression of the JSRV env gene is sufficient to transform an avian embryo fibroblast cell line, DF-1. DF-1 cells transfected with an avian sarcoma-leukaemia retroviral expression vector containing the JSRV env gene [pRCASBP(A)-J:env] exhibited changes consistent with transformation, including contraction and rounding of cells with formation of dense foci. Transfection with a reporter construct expressing the green fluorescent protein did not induce morphological changes in DF-1 cells, eliminating the possibility that the vector, the transfection protocol or culturing techniques were responsible for the transformed phenotype. When pRCASBP(A)-J:env-transfected cells were inoculated into nude mice, tumours formed, verifying that the DF-1 cells were tumorigenic. Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.
Allen
Thomas E
TE
Departments of Pathology1 and Microbiology2, Colorado State University, Fort Collins, CO 80523, USA.
Sherrill
Kate J
KJ
Departments of Pathology1 and Microbiology2, Colorado State University, Fort Collins, CO 80523, USA.
Crispell
Sara M
SM
Departments of Pathology1 and Microbiology2, Colorado State University, Fort Collins, CO 80523, USA.
Perrott
Matthew R
MR
Departments of Pathology1 and Microbiology2, Colorado State University, Fort Collins, CO 80523, USA.
Carlson
Jonathan O
JO
Departments of Pathology1 and Microbiology2, Colorado State University, Fort Collins, CO 80523, USA.
DeMartini
James C
JC
Departments of Pathology1 and Microbiology2, Colorado State University, Fort Collins, CO 80523, USA.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D005347:Fibroblasts / Q000166:cytology; D015752:Genes, env / Q000502:physiology*; D022141:Jaagsiekte sheep retrovirus / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011648:Pulmonary Adenomatosis, Ovine / Q000821:virology; D014162:Transfection; D014170:Transformation, Genetic; D018909:src Homology Domains