Cytoplasmic localization of the transforming protein of Fujinami sarcoma virus: salt-sensitive association with subcellular components

R A Feldman, E Wang, H Hanafusa
1983 Journal of Virology  
Fujinami sarcoma virus (FSV) encodes a transforming protein of 130,000 daltons (P130) which is associated with a tyrosine-specific protein kinase activity. To elucidate mechanisms involved in cell transformation by FSV, we have studied the intracellular location of P130 in rat cells nonproductively infected with FSV. Immunofluorescent staining of several FSV-transformed rat cell lines with a tumor regressor antiserum specific against the fps sequences of P130 showed that the major staining was
more » ... ocalized in the cytoplasm. Staining was also seen in cell ruffles and in some cases at areas of cell contact. The cytoplasmic location of P130 staining in cells infected with temperature-sensitive mutants of FSV was unchanged when they were grown at permissive or nonpermissive temperature. Cell fractionation of FSV-transformed cells under various conditions showed that the ionic strength used during cell fractionation had a striking effect on the distribution of P130. At 10 mM NaCl, 70% of P130 sedimented in the large granule fraction, whereas at 500 mM NaCl 70 to 90% of P130 was recovered in the cytosol fraction. Furthermore, a combination of ionic and nonionic detergents that effectively solubilized subcellular membranes was insufficient to solubilize P130 unless the salt concentration was raised. We conclude that the majority of P130 and its associated protein kinase activity are localized in the cytoplasm and that P130 is not an integral membrane protein. Fujinami sarcoma virus (FSV) is a defective avian sarcoma virus that causes rapid transformation both in vivo and in vitro (17, 24). The genome of FSV consists of viral gag sequences fused to unique, cell-derived sequences called fps. The gag-fps genome of FSV encodes a transforming protein of 130,000 daltons (P130) that contains gag-coded sequences at its N terminus andfps-coded sequences at its C terminus (17, 24). P130 is associated with a distinct tyrosine-specific protein kinase activity (13, 30) which has been implicated as essential to the mechanism of cell transformation by FSV (16, 30) . A cellular homolog of P130 has recently been identified by immunoprecipitation with antibody directed against fps-coded sequences. This protein, NCP98, has been shown to be antigenically, structurally, and enzymatically related to FSV P130 (26). It is believed that interaction of P130 with subcellular targets as yet unidentified triggers the process of cellular alteration to the transformed state. As a first step to identify these subcellular targets of P130 and to elucidate the mechanism of transformation by FSV, we undertook a study to determine the subcellular location of P130 and the nature of its association with subcellular components. These questions were examined by a combination of two different approaches: (i) indirect immunofluorescent staining, using a specific antibody directed against P130; and (ii) quantitative cell fractionation. To simplify the analysis, we chose rat fibroblasts transformed by FSV, because in these cells the only detectable viral protein being expressed is P130. The results of our study appear to indicate that P130 is primarily a cytoplasmic protein, but also present at some areas of the plasma membrane, and that it is not an integral membrane protein. MATERIALS AND METHODS Cells and viruses. Fisher rat embryo fibroblasts, 3Y1, infected with wild-type FSV were maintained as described previously (26). 3Y1 cells transformed with Schmidt-Ruppin Rous sarcoma virus (RSV), subgroup A (19), were provided by S. Kawai, University of Tokyo. 3Y1 cells were also transformed by temperature-sensitive FSV mutants, NY225 and NY240 (16), and clonal lines were selected for each transformant. The permissive and nonpermissive temperatures for these cultures were 32 and 38°C, respectively. The preparation of chicken embryo fibroblasts and their infection with FSV were described previously (17) . 782 on May 10, 2020 by guest
doi:10.1128/jvi.45.2.782-791.1983 fatcat:fharpnyupvh7lcaqryy54fq6vq