Fig 2. DENV replication is restricted by TRIM69.
(A) Cell viability assays of transient transfections of TRIM69 or vector. (B-E) DENV replication was restricted by TRIM69 ectopic expression. DENV RNA (B) and NS proteins (C) expression were analyzed in DENV-infected 293T cells with or without TRIM69 overexpression. (D) IF analysis of DENV proteins in Hela cells. Hela cells were transfected with or without TRIM69-Myc for 24 h, then infected with DENV-2 for another 24 h. NS3/NS4B rabbit Ab and Myc mouse Ab were used to perform the IF assays. (E) The titers of DENV from supernatants of infected 293T cells with or without TRIM69-Flag, as determined by TCID50 assays. (F) DENV DGL2 replicon replication was inhibited by TRIM69 ectopic expression. The supernatants from the cells were collected at indicated time-points post transfection to test the luciferase activity. (G-I) DENV-2 infection of normal (shNC) or TRIM69 knockdown (sh69-1 and sh69-2) 293T cells. (G) TRIM69 knockdown by shRNA did not cause cell toxicity. (H) NS3 and NS4B expression in TRIM69 silenced or control cells as tested by western blot. (I) Virus titers of DENV-2 from the supernatants of normal or TRIM69 knockdown cells. (J) DGL2 replicon replication in TRIM69 knockout 293T cells. TRIM69-/- cell line was generated by CRISPER/Cas9 system. (K) DGL2 replicon replication in Huh7.0 cells with TRIM69 knockdown. (L) The inhibitory efficiency of IFN-β on DGL-2 replication in normal or TRIM69 knockdown 293T cells (left). The TRIM69 mRNA levels in these cells were indicated by qRT-PCR (right). Results are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. The data shown are representative of at least 3 independent experiments.