A MicroRNA Screen Identifies the Wnt Signaling Pathway as a Regulator of the Interferon Response during Flavivirus Infection

Abstract

The impact of mosquito-borne flavivirus infections worldwide is significant, and many critical aspects of these viruses' biology, including virus-host interactions, host cell requirements for replication, and how virus-host interactions impact pathology, remain to be fully understood. The recent reemergence and spread of flaviviruses, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV), highlight the importance of performing basic research on this important group of pathogens. MicroRNAs (miRNAs) are small, noncoding RNAs that modulate gene expression posttranscriptionally and have been demonstrated to regulate a broad range of cellular processes. Our research is focused on identifying pro- and antiflaviviral miRNAs as a means of characterizing cellular pathways that support or limit viral replication. We have screened a library of known human miRNA mimics for their effect on the replication of three flaviviruses, DENV, WNV, and Japanese encephalitis virus (JEV), using a high-content immunofluorescence screen. Several families of miRNAs were identified as inhibiting multiple flaviviruses, including the miRNA miR-34, miR-15, and miR-517 families. Members of the miR-34 family, which have been extensively characterized for their ability to repress Wnt/β-catenin signaling, demonstrated strong antiflaviviral effects, and this inhibitory activity extended to other viruses, including ZIKV, alphaviruses, and herpesviruses. Previous research suggested a possible link between the Wnt and type I interferon (IFN) signaling pathways. Therefore, we investigated the role of type I IFN induction in the antiviral effects of the miR-34 family and confirmed that these miRNAs potentiate interferon regulatory factor 3 (IRF3) phosphorylation and translocation to the nucleus, the induction of IFN-responsive genes, and the release of type I IFN from transfected cells. We further demonstrate that the intersection between the Wnt and IFN signaling pathways occurs at the point of glycogen synthase kinase 3β (GSK3β)-TANK-binding kinase 1 (TBK1) binding, inducing TBK1 to phosphorylate IRF3 and initiate downstream IFN signaling. In this way, we have identified a novel cellular signaling network with a critical role in regulating the replication of multiple virus families. These findings highlight the opportunities for using miRNAs as tools to discover and characterize unique cellular factors involved in supporting or limiting virus replication, opening up new avenues for antiviral research.IMPORTANCE MicroRNAs are a class of small regulatory RNAs that modulate cellular processes through the posttranscriptional repression of multiple transcripts. We hypothesized that individual miRNAs may be capable of inhibiting viral replication through their effects on host proteins or pathways. To test this, we performed a high-content screen for miRNAs that inhibit the replication of three medically relevant members of the flavivirus family: West Nile virus, Japanese encephalitis virus, and dengue virus 2. The results of this screen identify multiple miRNAs that inhibit one or more of these viruses. Extensive follow-up on members of the miR-34 family of miRNAs, which are active against all three viruses as well as the closely related Zika virus, demonstrated that miR-34 functions through increasing the infected cell's ability to respond to infection through the interferon-based innate immune pathway. Our results not only add to the knowledge of how viruses interact with cellular pathways but also provide a basis for more extensive data mining by providing a comprehensive list of miRNAs capable of inhibiting flavivirus replication. Finally, the miRNAs themselves or cellular pathways identified as modulating virus infection may prove to be novel candidates for the development of therapeutic interventions.

Keywords: Wnt signaling; flavivirus; interferons; microRNA.

FIG 1

High-content microRNA screen against three flaviviruses, DENV, WNV, and JEV. (A) HeLa cells were transfected with miRNAs from the Dharmacon/Thermo Scientific v16.0 library and infected with DENV, WNV, or JEV (MOI = 0.5 FFU/cell) at 48 h posttransfection in 96-well plates in triplicate. At 24 h (WNV and JEV) or 48 h (DENV) posttransfection, cells were fixed and immunostained with antiflavivirus envelope antibody/AF488-anti-mouse IgG and DAPI to visualize nuclei. Total AF488 and DAPI signals were quantitated and normalized to those for miRNA control-transfected wells. miRNAs causing >20% toxicity were eliminated from the analysis. Volcano plots indicate the performance of miRNAs against DENV, WNV, and JEV infections. Active miRNAs (>60% inhibition; P value of <0.05) are denoted in blue. Highly represented miRNA families with activity against all three flaviviruses are shown at the right. (B) HeLa cells were reverse transfected with the indicated miRNAs and infected with DENV, WNV, and JEV at an MOI of 0.5 FFU/cell at 48 h posttransfection. Supernatants were collected at 48 h, and infectious virus was quantitated by a focus-forming assay.

FIG 2

miR-34 family members are broadly acting antiviral miRNAs. (A) HeLa cells were reverse transfected with a control miRNA (miR-Ct) or miR-34a and infected with DENV, WNV, and JEV at an MOI of 0.1 FFU/cell at 48 h posttransfection. Supernatants were collected at the indicated times postinfection and assayed for viral titers by a focus-forming assay. (B) ZIKV (strain PRVABC59) infections were carried out as described above for panel A at an MOI of 5 FFU/cell. (C) HFFs were transfected with miR-Ct or miR-34a and infected with WNV at an MOI of 3 FFU/cell at 48 h posttransfection. (D) Primary BM-DCs were electroporated with miR-Ct or miR-34a as described in Materials and Methods. At 48 h postelectroporation, cells were infected with WNV at an MOI of 0.01 FFU/cell. (E) HeLa cells were transfected with control or miR-34a LNAs and then infected with DENV at an MOI of 0.5 particles/cell. Supernatants were collected at the indicated times postinfection and assayed by a focus-forming assay. (F) HeLa cells were mock treated, infected with DENV at an MOI of 5 FFU/cell, or transfected with miR-Ct/miR-34a. At the indicated times postinfection or at 48 h posttransfection, total RNA was isolated, and the miR-34a expression level was quantitated by RT-qPCR (normalized to the U6 snRNP small RNA). (G) HeLa cells transfected with miR-Ct or miR-34a were infected with CHIKV, SINV, HSV-1, or VACV at an MOI of 0.5 particles/cell. Supernatants were collected at 24 to 48 h postinfection, and infectious units were quantitated by a plaque assay (CHIKV, HSV-1, and VACV) or a TCID₅₀ assay (SINV) (results are representative of data from >3 independent experiments) (*, P value of <0.05; **, P value of <0.01; ***, P value of <0.005). (H) HeLa cells were transfected with miR-34 family members (miR-34a/c and miR-449a/b), miR-Ct, or miR-34b and infected with DENV at an MOI of 5 FFU/cell at 48 h posttransfection. Cells were fixed at 48 h postinfection and immunostained with antienvelope/DAPI.

FIG 3

Repression of Wnt pathway factors by miR-34a dictates antiviral activity. (A) Total RNA isolated from miR-Ct- and miR-34a-transfected cells was assayed for transcript levels of the indicated Wnt pathway factors by RT-qPCR (*, P value of <0.05; **, P value of <0.01; ***, P value of <0.005). (B) HeLa cells were transfected with miR-Ct or miR-34a, followed by transfection 24 h later with pTOP-FLASH, a plasmid that expresses luciferase under the control of a Wnt-responsive promoter. Cells were treated at 24 h post-plasmid transfection with 2.5 μM and 25 μM Wnt agonist II to induce the Wnt/β-catenin pathway for 24 h. The luciferase level was quantitated by using the OneGlo luciferase system. (C) HeLa cells were transfected with siRNAs targeting the indicated transcripts or miR-Ct and -34a for 48 h, followed by infection with DENV at an MOI of 0.1 FFU/cell. Quantitation of infectious virus in supernatants collected at 3 days postinfection was performed by a focus-forming assay. (D) HeLa cells were transfected with the indicated siRNAs for 48 h. Total RNA was assayed for transcript levels of the indicated Wnt pathway factors by RT-qPCR.

FIG 4

miR-34a transfection potentiates type I interferon signaling. (A) miR-Ct/-34a-transfected HeLa cells were treated with the indicated stimuli [DENV at an MOI of 5 FFU/cell for 30 h, poly(I:C) transfected at 5 μg/ml for 18 h, and SeV at a 1:1,000 dilution for 6 h], and total RNA was isolated. Relative ISG56 levels were quantified by RT-qPCR and normalized to β-actin levels (*, P value of <0.01; **, P value of <0.005; ***, P value of <0.001; ns, not significant). (B) miR-Ct/-34a-transfected cells were treated with SeV for the indicated times, and total protein was analyzed by Western blotting for phosphorylated and total IRF3 levels. Results are representative of data from at least three independent experiments. Fold changes were calculated by measuring the integrated density by using ImageJ software and normalized to values for the control. (C) miR-Ct/-34a-transfected cells were treated with SeV for 6 h. Cytoplasmic (C) and nuclear (N) fractions were collected and analyzed by Western blotting for total IRF3, p84 (nuclear marker), and GAPDH (cytoplasmic marker). Results are representative of data from at least three independent experiments. (D) Supernatants from miR-Ct/-34a-transfected, SeV-treated cells were collected at 6 h and placed onto ISRE-luciferase-expressing telomerized human fibroblasts. At 18 h posttreatment, cells were lysed, and luciferase levels were quantified by using the OneGlo assay reagent. (E) HeLa cells were treated with SeV for 18 h. Total RNA was isolated and assayed for the Wnt-responsive transcript LEF1 or β-actin by TaqMan RT-qPCR. (Left) Fold changes were calculated by using the ΔΔC_T method. HeLa cells were transfected with the pTOP-FLASH reporter plasmid and then treated at 48 h posttransfection with Wnt agonist II or SeV. (Right) Cells were lysed and assayed for luciferase activity at 18 h posttreatment.

FIG 5

Repression of flavivirus infection occurs through IRF3-mediated interferon signaling. (A) miR-Ct/-34a- or siRNA-transfected cells were treated with SeV for 6 h. Total RNA was analyzed for ISG56 levels as described in the text. (B) Parental and IRF3KO HeLa cells were transfected with miR-Ct/-34a, followed by infection with DENV at an MOI of 5 FFU/cell. Supernatants were collected at 48 h, and infectious virus quantified by a focus-forming assay. WT, wild type. (C) Confirmation of IRF3 knockout by Western blotting (inset) and reactivity to SeV treatment by ISG56 RT-qPCR.

FIG 6

Interferon and Wnt pathways intersect through direct GSK3β/TBK1 interaction. (A) miR-Ct/-34a-transfected HeLa cells were mock or SeV treated for 6 h. Total protein was assessed for phosphorylated TBK1 and β-actin levels by Western blotting. Results are representative of data from at least three independent experiments. (B) miR-Ct/-34a-transfected cells were transfected with a plasmid encoding Flag-tagged TBK1 at 24 h post-miRNA transfection. At 24 h post-plasmid transfection, cells were treated with SeV for 6 h, lysed, and subjected to immunoprecipitation with Flag-Sepharose beads. Immunoprecipitated and total fractions were analyzed by Western blotting for GSK3β levels. IB, immunoblotting. (C) miR-Ct/-34a-transfected cells were treated with SeV as described above, with or without the GSK3β inhibitor SB216763 (5 μM). Phosphorylated and total IRF3 levels were assessed by Western blotting. Results are representative of data from at least three independent experiments. Fold changes were calculated by measuring the integrated density using ImageJ software and normalized to the values for the control.

FIG 7

Model of the Wnt-IFN signaling intersection. Innate activation by viral infection or dsRNA treatment (via Toll-like or RIG-I-like receptors) induces TBK1 phosphorylation (a), subsequent IRF3 phosphorylation/homodimerization (b), and translocation into the nucleus, where it induces the transcription of type I IFNs and interferon-stimulated genes (c). Simultaneous activation of the Wnt signaling pathway by viral infection (d) culminates in the repression of GSK3b phosphorylation (e), which feeds back positively into the IFN signaling pathway through interactions with TBK1 (f). The intersection between these two pathways suggests that the Wnt pathway can function to modulate the host inflammatory response as a way of controlling innate signaling. Inhibition of Wnt signaling by miR-34 (g) results in enhanced signaling through IRF3, promoting an antiviral state in the cell. IKK, IκB kinase.