DENV RNA replication triggers RIG-I and MDA5.

<p>IFN-β mRNA expression 24 hours post infection (h.p.i.) of DCs that were infected with DENV (A,B,F,K) after MAVS, TRIF, MYD88 (A), RIG-I, MDA5 (B), TBK1, IKKε (F) or IRF3 (K) silencing by RNA interference was measured by real-time PCR, normalized to GAPDH and set as 1 in DENV-infected DCs treated with control siRNA. (C,D) Flow cytometry analyses of TBK1 or IKKε phosphorylation at Ser172 in mock-treated or DENV-infected DCs 14 h.p.i. using phospho-specific antibodies. Data in (D) show relative mean fluorescent intensity (MFI). (E) Immunoblot of whole cell lysates of DCs mock-treated or infected with DENV in the presence or absence of DENV replication inhibitor SDM25N for TBK1 p-S172 and IKKε p-S172. Total TBK1 or IKKε served as loading control. (G) IFN-β mRNA of DENV-infected DCs 24 h.p.i. in the presence or absence of TBK1/IKKε inhibitor BX795 was measured by real-time PCR, normalized to GAPDH and set as 1 in DENV-infected DCs. (H) Confocal imaging of DNA (Hoechst, blue), DENV Envelope protein (E protein, green) or IRF3 (red) in mock-treated or DENV-infected DCs 18 h.p.i. White arrowheads indicate cells with nuclear IRF3. (I) Immunoblot for IRF3 of nuclear (NE) and cytoplasmic extracts (CE) of DCs infected with DENV at indicated times. β-actin served as loading control. (J) Similar as in (I) but IRF3 levels in nuclear extracts were measured by ELISA. Data are representative of at least five (H), four (C) or two (E,I) independent experiments with different donors or are collated (mean ± s.d.) of at least six (K), five (A) or four (B,D,F), three (G) or two (J) different donors. ** <i>P</i><0.01, * <i>P</i><0.05 (student’s t-test).