ZFYVE1 negatively regulates MDA5- but not RIG-I-mediated innate antiviral response
Fig 5
ZFYVE1 competes with MDA5 for viral RNA binding.
(A) ZFYVE1 inhibits the binding of MDA5 to poly(I:C)-HMW. HEK293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each). The lysates were incubated with biotinylated-poly(I:C)-HMW or biotinylated-5'ppp-dsRNA at 37°C for 1 h, and then incubated with streptavidin agarose at 37°C for another 2 h. The RNA-binding proteins were analyzed by immunoblots with anti-Flag and anti-HA. (B) ZFYVE1-deficiency enhances the binding of MDA5 to poly(I:C)-HMW. ZFYVE1-KO and control HEK293 cells (2 × 106) were transfected with an expression plasmid for Flag-MDA5 or HA-RIG-I (5 μg each). Twenty hours after transfection, cells were collected for pull-down assays similarly as in (A). (C) ZFYVE1 and MDA5 but not RIG-I bind to EMCV RNA. HEK293 cells were transfected with the indicated HA-tagged plasmids (5 μg each) for 20 h, then infected with EMCV for 1 h, washed with medium and cultured at 37°C for additional 2 h. Cell lysates were immunoprecipitated with IgG or anti-HA (5 μg) and protein G beads (50 μl) at 4°C for 2 h. The immunoprecipitates were treated with diluted RNase I (1:25 in PBS) at 37°C for 5 min. The bead-bound immunoprecipitates were washed for three times with lysis buffer containing RNase inhibitors. The protein and RNA complexes were eluted with 200 μl TE buffer containing 10 mM DTT at 37°C for 30 min. The protein-bound RNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of EMCV genome. Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the EMCV genome. (D) ZFYVE1, MDA5 and RIG-I bind to SeV RNA. HEK293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each) for 20 h, and then the cells were infected with SeV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). Positive (+) and negative (-) detections were shown at the top of the schematic presentation of the SeV genome. (E) ZFYVE1-deficiency enhances the binding of MDA5 to EMCV RNA. ZFYVE1-KO and control HEK293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each). At 20 h after transfection, cells were infected with EMCV for 1 h. Cell lysates were collected for “footprint” experiments similarly as in (C). (F) The C-terminal FYVE domain of ZFYVE1 is important for inhibition of MDA5 binding to poly(I:C)-HMW. HEK293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Eighteen hours later, cells were collected for in vitro pull-down assays similarly as in (A). (G) Overexpression of ZFYVE1 and its mutants inhibit EMCV-triggered activation of the IFN-β promoter. HT1080 cells were transfected with the IFN-β promoter reporter (0.2 μg) and ZFYVE1 or its mutant plasmids (0.2 μg) for 24 h, and then left un-infected or infected with EMCV for 12 h before luciferase assays. *P < 0.05 and **P < 0.01 (unpaired t test). Data shown are mean ± SD, n = 3 (technical replicate, C-E and G) and representative of three biological replicates (A-G) with similar results.