Fig 4. The E3 ligase activity and C-terminal portion are both critical for the anti-ZIKV action of TRIM56.

(A) Schematic representation of TRIM56 domains and the individual TRIM56 mutants investigated in this study. (B) HeLa-FitA2 cells with Dox-inducible expression of HA-T56-WT, HA-T56-Mut N, or HA-T56-AA were infected by ZIKV MR766 strain (MOI = 0.5) in the presence or absence of Dox for 72 h. The expression of ZIKV E, actin (loading control), and HA-tagged TRIM56 proteins were determined by immunoblotting. (C and D) HEK293 cells stably transduced with empty vector (Bsr), or Flag-T56-WT, Flag-T56-Mut N or Flag-T56-AA were infected by ZIKV MR766 strain for 72 h. Intracellular expressions of ZIKV E, actin (loading control), and Flag-tagged TRIM56 proteins were determined by immunoblotting (C), and progeny virus production in culture supernatants was measured by TCID₅₀ assay (D). Results shown in bar graphs were analyzed by one-way ANOVA test and reproduced for three times, **P<0.01. The immunoblotting data were representative of three independent experiments.