IFNα-dependent induction of endogenous BST-2 restricts MMTV release. GR cells were treated with 1000 units of endotoxin free recombinant IFNα or PBS for 24 hours. Virus was purified from culture supernatants and the corresponding cell extracts were harvested. (A) Cell extracts were used to determine level of BST-2 mRNA by qPCR, presented as fold change in BST-2 mRNA relative to vehicle treated cells; (B) and surface BST-2 by flow cytometry with the peak on the left as the isotype antibody control. Culture supernatants and their corresponding cell extracts were used to evaluate level of viral RNA, (C) extracellular viral RNA, (D) intracellular viral RNA, (E) and viral protein detected by antibody to MMTV capsid protein p27. Tubulin is a loading control. Data are presented as fold change in vRNA relative to vehicle treated samples. (F) Culture supernatants from IFNα or vehicle treated GR cells were used to infect TRH3 cells. Twenty four hours after infection, cells were harvested and used for DNA extraction followed by qPCR analysis of viral DNA normalized to GAPDH. Data are presented as fold change in viral DNA of cells infected with supernatants from IFNα treated cells relative to viral DNA of cells infected with supernatants from vehicle treated cells. Error bars are standard deviation, and p is the significance level, ns is not significant. Experiments were performed at least three times with similar results. (G) GR cells were stably transduced with shRNA targeting mouse BST-2 gene (shRNA) or a non-targeting shRNA (shControl). Cells were treated with IFNα (1000 units/ml) or vehicle. Twenty four hours later a portion of cells were used for FACS analysis of surface BST-2 level. (H) Culture supernatant and the remaining cells were examined for level of extracellular and intracellular viral proteins by Western blot.