Fig 1: ZIKV NS4B interacts with DHCR7 to inhibit TBK1 and IRF3 phosphorylation. A HEK293T cells were co-transfected with plasmids encoding HA-DHCR7, and plasmids encoding ZIKV non-structural proteins (FLAG-NS1, FLAG-NS3, FLAG-NS4A, FLAG-NS4B, and FLAG-NS5) or empty control plasmid for 30 ?h. The cell lysates were immunoprecipitated with anti-Flag antibody, and then analyzed by immunoblotting with indicated antibodies. B, C HEK293T cells were co-transfected with plasmids encoding HA-DHCR7, plasmids encoding FLAG-NS4B or empty control plasmid for 30 ?h. The cell lysates were immunoprecipitated with anti-HA antibody (B) or anti-FLAG antibody (C), and then analyzed by immunoblotting with indicated antibodies. D U251 ?cells were transfected with FLAG-NS4B plasmid together with plasmids encoding with HA-DHCR7. The localization of FLAG-NS4B (green), HA-DHCR7 (red), nucleus marker DAPI (blue), and merge were analyzed with confocal microscopy. Scale bar ?= ?10 ?µm. E Cell lysates from HEK293T cells transfected with HA-DHCR7 were incubated with GST protein or GST-NS4B protein which was incubated with glutathione-Sepharose beads. Mixtures were detected by Western blot with anti-HA and anti-GST antibody (top). Lysates from transfected HEK293T cells and the purified proteins were a detected by Western blot with anti-HA and anti-GST antibody (bottom). F U251 ?cells were transfected with plasmid encoding FLAG-NS4B (0, 1, 2 ?µg). The relative protein levels of DHCR7, FLAG-NS4B and GAPDH were detected by Western blot. G, H HEK293T cells were co-transfected with NS4B-expressing plasmid or empty vector, together with TBK1-coding plasmid (F), IRF3-expressing plasmid and plasmids encoding RIG-I (G). Cells were harvested at 24 ?h post-transfection and subjected to Western blot with indicated antibodies [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3(S396), anti-FLAG, and anti-GAPDH]. I DHCR7-silencing U251 (sh-DHCR7) and sh-NTC U251 ?cells were transfected with NS4B-expressing plasmid or empty vector. Cells were harvested at 6 ?h post-transfection and subjected to Western blot with indicated antibodies [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3(S396), anti-FLAG, and anti-GAPDH]. Data are representative of three independent experiments.