Fig. 6: Mutations at residues 67E/101P abolish the binding between ZIKV-NS2A and CYP17A1 mRNA and the inhibition of CYP17A1 translation.

a A model of ZIKV NS2A membrane topology⁴⁷. b Schematic representation of C-terminally truncated ZIKV-NS2A. c, d HEK293T cells were co-transfected with plasmids encoding hCYP17A1-HA/mCYP17A1-HA and each truncated NS2A-Flag. After 36 h post-transfection, the expression of h/mCYP17A1 and NS2A were detected by Western blotting. e Schematic representation of distinct NS2A mutants. f, g HEK293T cells were co-transfected with plasmids encoding hCYP17A1-HA/mCYP17A1-HA and distinct NS2A-Flag mutants. After 36 h post-transfection, the expression of h/mCYP17A1 and NS2A were detected by Western blotting. h Binding of distinct NS2A mutants with hCYP17A1 mRNA were analyzed by RIP-qPCR, and statistical significance was determined by comparing the results of each mutant protein group to the WT group. i hCYP17A1 mRNA-biotin was incubated with distinct mutant forms of GST-NS2A. After pulldown, the amounts of GST-NS2A in the input and eluates was detected by Western blotting. j, k hCYP17A1 mRNA was introduced into rabbit reticulocyte lysates along with distinct mutant forms of GST-NS2A or GST, and Lysates without hCYP17A1 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 hCYP17A1 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 c, d, f, g, i, j are representative of three independent experiments. The data shown in h, k are the mean ± s.d (h: n = 2; k: n = 3); Statistical analysis of (h, k) was performed using one-way ANOVA, followed by Tukey’s multiple comparison test. Source data are provided in the Source Data file.