Dengue virus subverts the interferon induction pathway via NS2B/3 protease-IκB kinase epsilon interaction

Abstract

Dengue is the world's most common mosquito-borne viral infection and a leading cause of morbidity throughout the tropics and subtropics. Viruses are known to evade the establishment of an antiviral state by regulating the activation of interferon regulatory factor 3 (IRF3), a critical transcription factor in the alpha/beta interferon induction pathway. Here, we show that dengue virus (DENV) circumvents the induction of the retinoic acid-inducible gene I-like receptor (RLR) pathway during infection by blocking serine 386 phosphorylation and nuclear translocation of IRF3. This effect is associated with the expression of nonstructural 2B/3 protein (NS2B/3) protease in human cells. Using interaction assays, we found that NS2B/3 interacts with the cellular IκB kinase ε (IKKε). Docking computational analysis revealed that in this interaction, NS2B/3 masks the kinase domain of IKKε and potentially affects its functionality. This observation is supported by the DENV-associated inhibition of the kinase activity of IKKε. Our data identify IKKε as a novel target of DENV NS2B/3 protease.

FIG 1

Interferon regulatory factor 3 (IRF3) phosphorylation state at residue S386 after infection and/or stimulation of TLR3 or RLR pathways. (A) Levels of phosphorylated IRF3 (pS386-IRF3) and IRF3 on a Western blot prepared with 20 μg of whole-cell extract recovered from cells treated as indicated and collected at 24 h postinfection and 12 h poststimulation. GAPDH was detected in these same cell lysates as a loading control and DENV NS5 protein as evidence of viral replication. (B) Densitometric analysis of pS386-IRF3 protein levels normalized to GAPDH are shown in arbitrary units. **, significant difference between RLR-stimulated cells that were DENV infected and mock infected (P < 0.01, two-tailed Student's t test) (n = 3). Each bar represents the mean ± SEM. −C, negative control.

FIG 2

Cellular localization of IRF3 and pS386-IRF3 after DENV infection. The 293/TLR3 cells were infected or mock infected with DENV and stimulated or not for TLR3 or RLR as indicated. The cells were dually labeled for DENV and IRF3 (A to C, mock infected; D to F, DENV infected) or pS386-IRF3 (G to I, mock infected; J to L, DENV infected). Cells were stained with fluorescent, conjugated secondary antibodies and observed by confocal microscopy. Red indicates IRF3 or pS386-IRF3, green indicates DENV infection, and blue indicates nuclei stained with DAPI.

FIG 3

NS2B/3 inhibits RLR-associated induction of pS386-IRF3 phosphorylation. (A) The cells were transfected with NS2B/3- or NS2B/3-S135A-encoding plasmids, and 36 h after transfection, they were stimulated for RLR pathways for another 12 h and then cell lysate (20 μg) was analyzed by Western blotting using an anti-pS386-IRF3 antibody. (B) Densitometric analyses of pS386-IRF3 protein levels normalized to GAPDH are shown as fold change compared to the positive control (+C). *, significant difference compared to the +C (P < 0.05, one-way ANOVA) (n = 2). Each bar represents the mean ± SEM.

FIG 4

DENV protease interacts with human IKKε. (A) 293/TLR3 cells were transfected with HA-NS2B/3 or HA-NS2B/3-S135A and Flag-IKKε or empty plasmid and at 48 h posttransfection were evaluated by proximity ligation assay (PLA). Single detection of proteins as controls for expression: IRF3 (a), IKKε (b), HA-NS2B/3 (c), and HA-NS2B/3-S135A (d). Red fluorescence indicates positive expression of these proteins and blue indicates nuclear staining by DAPI. (e to j) Double detection of protein-protein interactions. HA-tagged proteins were stained with a primary anti-HA antibody and a secondary PLA Minus probe, and IKKε was stained using a primary anti-IKKε antibody and a secondary PLA Plus probe. When the proteins are in close proximity, fluorescence is generated. The absence of red fluorescence reflects no protein-protein interaction. Shown are the endogenous expression of IKKε (e and f) and IKKε overexpression (g and h). (B) Cells were lysed at 48 h posttransfection and extracts coimmunoprecipitated with antibody against HA, and the bound and unbound fractions were analyzed by immunoblotting for IKKε.

FIG 5

Analysis of IKKε putative cleavage by DENV protease NS2B/3 and IKKε functionality after DENV infection. (A) Representation of IKKε structure, including two putative target sites for NS2B/3 protease. (B and C) IKKε cleavage evaluation by Western blotting. (B) 293/TLR3 cells were transfected with Flag-IKKε- and NS2B/3- or NS2B/3-S135A-encoding plasmids for 48 h, and then the cell lysate (20 μg) was analyzed using an anti-Flag or anti-IKKε antibody. (C) Mock- or DENV-infected cells after 24 h postinfection analyzed for IKKε endogenous expression in 40 μg of whole-cell extract using an anti-IKKε antibody. (D) IKKε functionality evaluation. In the top blot, the cells were transfected with IKKε for 24 h, and then infected with DENV-2 for another 24 h to evaluate the ability of DENV to affect IKKε-associated phosphorylation of S386-IRF3. (E) Densitometric analysis of pS386-IRF3 protein levels. The results were normalized to GAPDH. The asterisks represent a significant fold change compared to IKKε-transfected cells using one-way ANOVA: *, P < 0.05; **, P < 0.01 (n = 2). Each bar represents the mean ± SEM.

FIG 6

Interaction model of human IKKε with the NS2B/3 protein complex. Shown are the molecular surfaces for human IKKε and NS2B/3 (PDB ID, 2FOM) separated and docked with a side view (upper panel) and a top view (lower panel). The yellow residues represent the IKKε protein kinase domain, residues in orange represent the ATP-binding region, and the remaining residues are shown in gray. Blue residues represent the NS2B/3 complex, and magenta represents the catalytic triad (His51, Asp5, and Ser135). The close-up panels (bottom) illustrate in red two important residues in the kinase domain, Lys38 (ATP-binding) and Asp135 (proton acceptor). The model was generated using the fully automatic ClusPro 2.0 protein-protein docking server and visualized using PyMOL.