Isolation of a transformation-defective mutant of the McDonough strain of feline sarcoma virus exhibiting tyrosine kinase activity in vitro but not in vivo.
NRK cells transformed by the McDonough strain of feline sarcoma virus (SM-FeSV) were mutagenized by the use of 5'-azacytidine. Four cell lines expressing different transformation-defective phenotypes were isolated. Superinfection of these cell lines with simian sarcoma-associated virus (SSAV) led in three instances to the recovery of transforming virus particles carrying an intact fms gene. A nonconditional transformation-defective virus, designated td26-SM-FeSV (SSAV), was isolated from one of the cell lines. NRK cells infected with this mutant contained actin cables and fibronectin networks and exhibited normal cell morphology. Such cells formed only small colonies in soft agar and exhibited a mitogenic activity similar to that of noninfected cells. Cells infected with td26-SM-FeSV (SSAV) synthesized a gag-fms fusion glycoprotein (gp180gag-fms). This polypeptide was processed in the normal manner into the intracellular gp120v-fms and a transformation-defective gp140td-v-fms which was expressed at the surface of infected cells. This species had an increased electrophoretic mobility on polyacrylamide gels compared with the molecule from wild-type virus.gp140td-v-fms had endo-beta-N-acetylglucosaminidase H-resistant carbohydrate side chains. No tyrosine kinase activity was detectable in vivo in td26-SM-FeSV (SSAV)-infected cells even when the cells were treated with sodium orthovanadate. In vitro, fms molecules from td26-SM-FeSV (SSAV)-infected cells exhibited tyrosine kinase activity as determined by autophosphorylation and phosphorylation of exogenous (poly)Glu-Tyr. At low ATP concentrations (less than 5 microM) this in vitro tyrosine kinase activity was significantly reduced compared with that of the wild-type counterpart.
Tamura
T
T
Institut für Medizinische Virologie, Justus-Liebig-Universität, Giessen, Federal Republic of Germany.
Hennig
D
D
Grell
M
M
Niemann
H
H
Boschek
B
B
D000954:Antigens, Surface / Q000032:analysis; D002460:Cell Line; D002472:Cell Transformation, Viral; D004252:DNA Mutational Analysis; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008970:Molecular Weight; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000502:physiology*; D009857:Oncogenes; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D005253:Sarcoma Viruses, Feline / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000235:genetics
D000954:Antigens, Surface; D009856:Oncogene Proteins, Viral; D010750:Phosphoproteins; D014760:Viral Fusion Proteins; D011505:Protein-Tyrosine Kinases