Figure 3.

RIG-I-associated RNA after VSV infection is immunostimulatory and contains base-paired RNAs with 5’-triphosphate modifications. HEK 293 cells (40 x 10⁶) expressing FLAG-RIG-I or not were infected with VSV (MOI=1) or left uninfected. 9 hours later lysates were prepared and either used directly for analysis by SDS-PAGE and RNA isolation or immunoprecipitated with anti-FLAG antibody-coupled sepharose beads or beads coupled to an unspecific IgG-isotype control. After elution of the protein/RNA complexes from the beads, co-immunoprecipitated RNA was purified from the eluate and further analyzed as indicated. Aliquots of the immunoprecipitated proteins were analyzed by SDS-PAGE. (A) Schematic representation of the experimental set-up. RD (regulatory or repressor domain) the domain of RIG-I binding to the 5’-end of triphosphate modified RNA; also called c-terminal domain (CTD). (B) RNA isolated directly from infected cells or after immunoprecipitation was retransfected into 1205Lu melanoma cells (8 x 10³ cells/well) using RNAiMax Lipofectamin. IP-10 was measured in the supernatant after 24 hours by ELISA. In the lower panel western blot results from the corresponding lysates are depicted after staining with anti-FLAG antibodies. (C) RNA was treated with the indicated enzymes prior to retransfection into 1205Lu melanoma cells. IP-10 was measured in the supernatant after 24 hours. Data are shown as mean +- SD from n=2 independent experiments.