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 IKKε. 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)