Dengue Virus Targets the Adaptor Protein MITA to Subvert Host Innate Immunity (original) (raw)
Figure 3
Mapping of the dengue protease cleavage site of MITA.
(A) Schematic diagram and summarized properties of MITA constructs. Constructs were N-terminal HA- and C-terminal V5-tagged and are numbered according to the amino acid residues. The potential cleavage site LRRG in human MITA and the corresponding sequence IHCM in murine MPYS are indicated. (B) A549 cells were transfected with the full-length or deletion constructs of MITA with or without the Flag-tagged dengue NS2B3. Transfectants were harvested for immunoblotting with antibodies indicated at the right. The positions for full-length MITA are indicated by black arrows and the cleaved MITA by open arrows. (C) A549 cells were cotransfected with Vip-Luc (0.2 µg), IRF3/pCR3.1 (0.3 µg), pRL-TK (0.1 µg), plus GFP control or the indicated MITA constructs (0.4 µg) for 24 h. The cells were harvested and analyzed by dual-luciferase assay. The relative normalized luciferase activities are expressed as mean and SD (n = 3 per group), and were compared by two-tailed Student's t test. (D) Immunoblotting of A549 cells cotransfected with DEN-2 NS2B3 plus the indicated constructs of MITA or MPYS for 24 h. (E) Dual-luciferase assay of A549 cells cotransfected with Vip-Luc (0.15 µg), IRF3/pCR3.1 (0.15 µg), pRL-TK (0.05 µg), plus the wild-type or S135A-mutated dengue NS2B3 (0.35 µg) with MITA or MPYS (0.3 µg) for 24 h. GFP was used as the negative control. The cells were harvested and analyzed by dual-luciferase assay. Data are expressed as mean and SD (n = 3 per group), and were compared by two-tailed Student's t test.