A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissibl e lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we hav e found that the expression of the JSRV envelope (Env) is sufficient to tra nsform mouse NIH 3T3 cells in classical transformation assays. To further i nvestigate the mechanisms of JSRV oncogenesis, we generated a series of env elope chimeras between JSRV and the JSRV-related endogenous retroviruses of sheep (enJSRVs) and assessed them in transformation assays. Chimeras conta ining the exogenous JSRV SU region and the enJSRV TM region were unable to transform NIH 3T3 cells. Additional chimeras containing only the carboxy-te rminal portion of TM (a region that we previously identified as VR3) of the endogenous envelope with SU and the remaining portion of TM from the exoge nous JSRV were also unable to transform NIH 3T3 cells. The VR3 region inclu des the putative membrane-spanning region and cytoplasmic tail of the JSRV TM glycoprotein; this suggested that the cytoplasmic tail of the JSRV Env m ediates transformation, possibly via a cell signaling mechanism. Mutations Y590 and M593 in the cytoplasmic tail of the JSRV envelope were sufficient to inhibit the transforming abilities of these constructs. Y590 and M593 ar e part of a Y-X-X-M motif that is recognized by the phosphatidylinositol 3- kinase (PI-3K). PI-3K initiates a cell signaling pathway that inhibits apop tosis and is required for a number of mitogens during the G(1)-to-S-phase t ransition of the cell cycle. PI-3K activates Akt by phosphorylation of thre onine 308 and serine 473. We detected by Western blot analysis phosphorylat ed Akt in serum-starved MP1 cells (NIH 3T3 cells transformed by JSRV) but n ot in the parental NIH 3T3 cells. These data indicate that the cytoplasmic tail of the JSRV TM is necessary for cell transformation and suggest a new mechanism of retroviral transformation. In addition, the ability to dissoci ate the function of the JSRV envelope to mediate viral entry from its trans forming capacity has direct relevance for the design of JSRV-based vectors that target the differentiated epithelial cells of the lungs.