Fig. 4: ZIKV-NS2A interacts with CYP17A1 mRNA and reduces its translation.

a HEK293T cells were co-transfected with plasmids encoding CYP17A1-HA and NS2A-Flag or empty vector as control. At 24 h post-transfection, cells were treated with 40 μg/mL CHX for 0, 3 h. The expression of CYP17A1 and NS2A were detected by Western blotting. b HEK293T cells were transfected with NS2A-Flag for 20 h, followed by a subsequent transfection with in vitro synthesized CYP17A1 mRNA for 4 h. The expression of CYP17A1 and NS2A were detected by Western blotting. c CYP17A1-A549 cells were infected with HA-NS2A ZIKV for 24 h. RIP-qPCR were performed to determine the binding between CYP17A1 mRNA and HA-NS2A. The levels of CYP17A1 and GAPDH mRNA was calculated by comparing the amount of RNA precipitated by specific antibody (viral E, claudin-2, or HA) with the amount of the same type of RNA precipitated by isotype control IgG. d MST workflow. See text for detail. e Binding affinities of GFP-NS2A to CYP17A1 mRNA were measured by MST as described in the methods. f The workflow of the in vitro RNA-protein pulldown assay. g The amounts of GST-NS2A in the input and eluates from the pulldown assay were detected by Western blotting. h Competition assay was used to determine the binding affinity between CYP17A1 mRNA-biotin and GST-NS2A in the presence of increasing amounts of competitors. After pulldown, GST-NS2A in the eluates was detected by Western blotting. i EMSA was used to determine the binding affinity of CYP17A1 mRNA-biotin with ZIKV-NS2A. CYP17A1 mRNA-biotin was incubated with increasing amounts of GST-NS2A, with GST used as a control. CYP17A1 mRNA-biotin migration was performed in 0.4% agarose gel without sodium dodecyl sulfate (SDS) and detected using Streptavidin-HRP Conjugate. j Quantification of ribosome loading on to CYP17A1 transcripts. CYP17A1-HA was co-transfected into HEK293T cells with empty vector, NS2A-Flag, and E-Flag, respectively. At 48 h post-transfection, cells were treated with 100 μg/mL CHX for 10 min and used to analyze both the total and ribosome-associated levels of GAPDH, CYP17A1 and Calnexin mRNA. k CYP17A1 mRNA was introduced into rabbit reticulocyte lysates along with increasing amounts of GST-NS2A or GST, and Lysates without CYP17A1 mRNA served as a blank control in the in vitro translation system. The mixtures were incubated at 30 °C for 90 min, and the expression of CYP17A1 in the lysates was detected by Western blotting. The bar plot shows quantification of the band intensity from the western blot image. The images in a, b, g–i, k are representative of three independent experiments. The data shown in c, j, k are the mean ± s.d (n = 3 independent experiments); Statistical analysis of (c, j, k) was performed using two-way ANOVA, followed by Tukey’s multiple comparison test (c, j); one-way ANOVA, followed by Tukey’s multiple comparison test (k); ns not significant. Source data are provided in the Source Data file.