Role of gag sequence in the biochemical properties and transforming activity of the avian sarcoma virus UR2-encoded gag-ros fusion protein.
The transforming protein P68gag-ros of avian sarcoma virus UR2 is a transmembrane tyrosine protein kinase molecule with the gag portion protruding extracellularly. To investigate the role of the gag moiety in the biochemical properties and biological functions of the P68gag-ros fusion protein, retroviruses containing the ros coding sequence of UR2 were constructed and analyzed. The gag-free ros protein was expressed from one of the mutant retroviruses at a level 10 to 50% of that of the wild-type UR2. However, the gag-free ros-containing viruses were not able to either transform chicken embryo fibroblasts or induce tumors in chickens. The specific tyrosine protein kinase activity of gag-free ros protein is about 10- to 20-fold reduced as judged by in vitro autophosphorylation. The gag-free ros protein is still capable of associating with membrane fractions including the plasma membrane, indicating that sequences essential for recognition and binding membranes must be located within ros. Upon passages of the gag-free mutants, transforming and tumorigenic variants occasionally emerged. The variants were found to have regained the gag sequence fused to the 5' end of the ros, apparently via recombination with the helper virus or through intramolecular recombination between ros and upstream gag sequences in the same virus construct. All three variants analyzed code for gag-ros fusion protein larger than 68 kDa. The gag-ros recombination junction of one of the transforming variants was sequenced and found to consist of a p19-p10-p27-ros fusion sequence. We conclude that the gag sequence is essential for the transforming activity of P68gag-ros but is not important for its membrane association.
Jong
S M
SM
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029-6574.
Wang
L H
LH
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002642:Chick Embryo; D003001:Cloning, Molecular; D004742:Enhancer Elements, Genetic; D015681:Fusion Proteins, gag-onc / Q000235:genetics*; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011533:Proviruses / Q000235:genetics; D015183:Restriction Mapping; D014162:Transfection
D015681:Fusion Proteins, gag-onc; D011505:Protein-Tyrosine Kinases