Fig 1: ZIKV NS4A interacts with both the CL and TM domains of MAVS and prevents RIG-I from binding MAVS. a A schematic diagram of the MAVS protein and functional domains: CARD-like domain (aa 10 to 77), proline-rich domain (aa 103 to 173) and transmembrane domain (aa 514 to 535). b Co-IP and western blotting analysis of 293T cells transfected with Myc-tagged NS4A along with vectors expressing the indicated Flag-tagged MAVS truncation forms or full-length MAVS. Empty vector was used as a negative control. c 293T cells were co-transfected with Flag-tagged RIG-I and Myc-tagged NS4A or an empty vector control for 24 h. Whole cell lysates were subjected to immunoprecipitation using an anti-MAVS antibody and analyzed by western blotting for RIG-I (c). 293T cells were transfected with Myc-tagged NS4A or an empty vector control for 24 h later, transfected with 20 µg/ml of poly(I:C). Whole cell lysates were subjected to immunoprecipitation using an anti-MAVS antibody and analyzed by western blotting for TRAF6 (d), or TBK1 (e). The expression of precipitated proteins was determined by western blotting analysis using the indicated antibodies (lower panel). The data shown are representative of three independent experiments with similar results
Fig 2: Molecular interaction between ZIKV NS4A protein and the type I IFN induction pathway. GAL4-based mammalian two-hybrid screening assays were performed to identify the molecular targets of ZIKV proteins in RIG-I signaling. 293T cells in 24-well plates were co-transfected with a pGL4.31 vector, a pFN11A (BIND) vector expressing a fusion protein of GAL4-BD and individual ZIKV prM, NS4A or DENV NS4A proteins, and a pFN10A (ACT) vector expressing a RIG-I, b MAVS, c TBK1 or d IKKe. The pFN11A (BIND) vector contained a Renilla luciferase gene that was used as an internal control to normalize DNA transfection efficiency. The pBIND and pACT vectors were used as negative controls, and the pBIND-Id and pACT-MyoD vectors were used as positive controls (PCs) according to the manufacturer’s instructions. At 48 h post-transfection, cell lysates were harvested for the luciferase activity assay. The results are shown as relative luciferase activity after normalization with Renilla luciferase activity. Data are shown as the mean ± SD derived from three repeat experiments. *p < 0.05, and **p < 0.01 (Student’s t test)
Fig 3: ZIKV NS4A co-localizes and interacts with MAVS. a HeLa cells transfected with plasmids expressing Flag-tagged NS4A, Flag-tagged ZIKV prM or influenza Flag-tagged PB1-F2 were stained with anti-Flag and anti-MAVS antibodies as well as DAPI. Secondary antibodies conjugated to rhodamine and FITC dye were used to visualize the indicated proteins. Images are representative of three independent experiments. 293T cells co-transfected with plasmids encoding Myc-tagged MAVS and Flag-tagged NS4A were used in a co-IP assay to address whether ZIKV NS4A protein physically interacts with MAVS. Cell lysates were precipitated with an anti-Flag antibody (b), anti-Myc antibody (c), or control mouse IgG, and immunocomplexes were analyzed with the indicated antibodies by western blotting. d 293T cells were transfected with plasmids encoding Flag-tagged NS4A, followed by immunoprecipitation using anti-Flag antibody or control IgG. The immunocomplexes were analyzed with anti-MAVS antibody by Western blotting. e HFF-1 cells were infected with ZIKV at an MOI of 5 followed by immunoprecipitation using anti-MAVS antibody or control mouse IgG. The immunocomplexes that were captured by the protein G Dynabeads were analyzed by Western blotting using anti-NS4A, or anti-MAVS antibodies. f SPR analysis of the interactions between MAVS and NS4A. Direct binding was measured by Biacore assays. MAVS was immobilized on a CM5 chip. The analytes consisted of serial dilutions of NS4A proteins ranging between 0 and 2000 nM. The data shown are representative of three independent experiments with similar results
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