Jaagsiekte sheep retrovirus proteins in oncogenic transformation and regulation of viral gene expression

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). It has been clearly established that the native envelope (Env) structural protein of JSRV has oncogenic potential and is the determinant of JSRV-mediated transformation. The subject of this dissertation was two-fold; to investigate the domains of Env that participate in JSRV-induced transformation and to describe a novel JSRV regulatory protein encoded within the env gene.;Results from transformation assays in NIH 3T3 fibroblast using chimeric Env proteins between endogenous and exogenous isolates of JSRV indicated that the cytoplasmic tail (CT) domain in the transmembrane (TM) region of Env is necessary for transformation. However, these experiments did not clarify if the TM domain alone is sufficient for transformation. Transformation assays using a series of deletion mutants through the surface (SU) domain of Env indicated that all of these mutants were unable to transform fibroblasts cells. These results were not due to inadequate expression or transport to the plasma membrane, as confirmed by Northern and Western blot anaysis, and immunofluorescence microscopy. These data indicated that domains in SU facilitate JSRV transformation, further supported by complementation between SU and TM mutants for transformation.;The Tetracysteine (TC) Tag-FlAsH(TM) system provides a sensitive method for in-vivo detection and subcellular localization of proteins fused to the TC-Tag using fluorescence microscopy. Generation of a JSRV env expression construct fused to the TC-tag induced transformation comparable to wild-type Env, and these tagged-proteins were successfully detected in transformed fibroblasts. These experiments lay the foundation for future studies aimed at enhanced imaging of wild-type and mutant JSRV Env proteins in live cells.;Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. In this dissertation, it was also demonstrated that JSRV encodes within the env gene a trans-acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences abolished JSRV to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by co-transfection of an Env expression plasmid. Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and an expression construct for the signal peptide showed Rej activity. Interestingly, JSRV expression plasmids deleted in Rej showed normal transport of unspliced JSRV RNA to the cytoplasm. Thus, unlike for other analogous retroviral regulatory proteins, the primary function of JSRV Rej is not to facilitate export of unspliced viral RNA to the cytoplasm. Its action might be to facilitate unspliced viral RNA translation.