Identification of Cellular Proteins Important for Jaagsiekte Sheep Retrovirus Transformation
Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a contagious lung cancer in sheep, ovine pulmonary adenocarcinoma (OPA). The envelope gene (env) also is an oncogene, since it induces cell transformation and tumors on its own. The subject of this thesis was to identify cellular proteins that interact with JSRV Env and to assess their roles in JSRV transformation. A previous yeast two-hybrid screen identified candidate proteins that can interact with the JSRV envelope protein. Two were studied here: Zinc Finger Protein 111 (Zfp111) and Ribonucleotide Reductase subunit 2 (RRM2).
For Zfp111, shRNA knockdown of endogenous zfp111 in rat 208F fibroblasts reduced transformation by JSRV Env but not by another viral oncogene v-mos. Env transformation was restored by a knockdown-resistant Zfp111 cDNA, and over-expression of zfp111 increased transformation by Env but not v-mos. Knockdown of zfp111 decreased proliferation rates of Env transformed cells but not untransformed cells.
Zfp111 bound to a smaller form of Env (P70env); while the Env polyprotein (Pr80env) is cytoplasmic, P70env is nuclear. P70env and Pr80env have the same polypeptide backbone, so they differ in glycosylation. Co-expression of Zfp111 with JSRV Env stabilizes both proteins. Selected alanine scanning mutants in the Env cytoplasmic tail (CT) were co-transfected with Zfp111; there was a strong correlation between mutant transformation efficiencies and levels of P70env and Zfp111 detected. The results suggested a putative interaction region for Zfp111 in the Env CT.
With regard to RRM2, endogenous RRM2 co-localized with Env by re-localization to the plasma membrane in transfected NIH 3T3 cells. In rat 208F cells, RRM2 knockdown decreased Env transformation, but there was also a decrease (significantly less) in v-mos transformation. RRM2 knockdown cells showed a decrease in overall growth rates, which might explain the effect on v-mos transformation.
Progress towards tandem affinity purification (TAP) of JSRV Env-associated cellular proteins is also described. JSRV Env with a C-terminal TAP tag (HBH) was generated. The HBH-tagged Env could transform cells, and it could be successfully purified over Ni2+ and streptavidin columns; JSRV Env peptide sequences (SU and TM) were identified in preliminary TAP/MS experiments.