Inhibition of miR-200b-3p confers broad-spectrum resistance to viral infection by targeting TBK1

ABSTRACT The host innate immune system’s defense against viral infections depends heavily on type I interferon (IFN-I) production. Research into the mechanisms of virus-host interactions is essential for developing novel antiviral therapies. In this study, we compared the effect of the five members of the microRNA-200 (miR-200) family on IFN-I production during viral infection and found that miR-200b-3p displayed the most pronounced regulatory effect. During viral infection, we discovered that the transcriptional level of microRNA-200b-3p (miR-200b-3p) increased with the infection of influenza virus (IAV) and vesicular stomatitis virus (VSV), and miR-200b-3p production was modulated by the activation of the ERK and p38 pathways. We identified cAMP response element binding protein (CREB) as a novel transcription factor that binds to the miR-200b-3p promoter. MiR-200b-3p reduces NF-κB and IRF3-mediated IFN-I production by targeting the 3′ untranslated region (3′ UTR) of TBK1 mRNA. Applying miR-200b-3p inhibitor enhances IFN-I production in IAV and VSV-infected mouse models, thus inhibiting viral replication and improving mouse survival ratio. Importantly, in addition to IAV and VSV, miR-200b-3p inhibitors exhibited potent antiviral effects against multiple pathogenic viruses threatening human health worldwide. Overall, our study suggests that miR-200b-3p might be a potential therapeutic target for broad-spectrum antiviral therapy. IMPORTANCE The innate immune response mediated by type I interferon (IFN-I) is essential for controlling viral replication. MicroRNAs (miRNAs) have been found to regulate the IFN signaling pathway. In this study, we describe a novel function of miRNA-200b-3p in negatively regulating IFN-I production during viral infection. miRNA-200b-3p was upregulated by the MAPK pathway activated by IAV and VSV infection. The binding of miRNA-200b-3p to the 3′ UTR of TBK1 mRNA reduced IFN-I activation mediated by IRF3 and NF-κB. Application of miR-200b-3p inhibitors exhibited potent antiviral effects against multiple RNA and DNA viruses. These results provide fresh insight into understanding the impact of miRNAs on host-virus interactions and reveal a potential therapeutic target for common antiviral intervention.

propagated in Vero cells. Sendai virus (SeV, Cantell strain, Charles River Laboratories) was propagated in chicken embryos and used at a final concentration of 100 hemagglutinin units per mL.
The JNK inhibitor (no. GC13841), the NF-kB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), Asperuloside (no. GC35411), and the CREB inhibitor (no. GC32689) were all purchased from Glpbio (Montclair, CA). All the experiments involving mice were performed following the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China and were approved by the Scientific Ethics Committee of Huazhong Agricultural University (permit number: HZAUMO-2017-056).

Virus titration
For IAV titration, viral titers of virus stocks and cell culture supernatants were deter mined by end-point titration in MDCK cells. Tenfold serial dilutions of each sample were inoculated into MDCK cells. Two days after inoculation, supernatant from the inoculated cells was collected and tested for the ability to agglutinate chicken erythrocytes as an indicator of viral replication. Infectious viral titers are reported as log 10 TCID 50 /mL and were calculated from three replicates by using the method of .
For RABV and VSV titration, N2a cells were infected with serial dilutions of the viruses. After 1 h incubation at 37°C, the cell supernatant was discarded and washed once with PBS, and then overlaid with DMEM containing 1% low melting point agarose (VWR, 2787C340). After incubation at 34°C for 72 h, 293T cells were stained with FITC-conju gated antiRABV N antibody (Fujirebio Diagnostics, Malvern, PA). Then, the fluorescent foci were counted under a fluorescence microscope. For VSV titration, the plaques were counted at 48 h postinfection.
For HSV-1 titration, Vero cells were seeded in 12-well plates and infected with serial dilutions of the viruses. After 1 h incubation at 37°C, the cell supernatant was discarded and washed once with PBS and then overlaid with DMEM containing 1% low melting point agarose. After incubation at 34°C for 48 h, the agarose was removed and then fixed and stained with a solution of 0.1% crystal violet and 10% formalin in PBS under UV light. After staining for 4 h, the plates were washed with water, and the plaques were counted.
For JEV titration, BHK-21 cells were seeded in 12-well plates and infected with serial dilutions of the viruses. After 1 h incubation at 37°C, the cell supernatant was discarded and washed once with PBS, and then overlaid with DMEM containing 1% low melting point agarose. After incubation at 34°C for 48 h, the agarose was removed and then fixed and stained with a solution of 0.1% crystal violet and 10% formalin in PBS under UV light. After staining for 4 h, the plates were washed with water, and the plaques were counted.

RNA isolation and quantitative real-time PCR (qPCR)
Total RNA was isolated from cells and tissues by using TRIzol reagent (Invitrogen). The genomic DNA was eliminated with TURBO DNA-free Kit (Invitrogen, AM1907) as per the manufacturer's instructions. RNA quality was assessed by using NanoDrop 2,000 (Thermo Scientific). The cDNAs were synthesized by ReverTra Ace qPCR RT Master Mix (Toyobo, FSQ-201) or First-Strand cDNA Synthesis Kit (Vazyme, R211-01). qPCR was performed using SYBR Green Supermix (Bio-Rad, 172-5124). Primers for qPCR are listed in Table 1 The method for stem-loop real-time quantification of miRNA was described previously (33), and the schematic diagram is shown in Fig. S10. For miRNA quantifica tion, total RNA from cells was purified using the MiPure Cell/Tissue miRNA Kit (Vazyme, RC201) to retain small RNA according to the manufacturer's protocol. The cDNAs were synthesized by miRNA 1st Strand cDNA Synthesis Kit (Vazyme, MR101-01). Stem-loop

Western blot
Cells were cultured in 24-well plates and lysed with NP40 lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40) supplemented with a protease inhibitor cocktail and PMSF (1 mM) for 30 min at 4°C. The cell lysates were centrifuged for 10 min at 12,000 g and 4°C. The supernatants were transferred into a new tube, resolved by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes (Bio-Rad) to measure protein expression. The membrane was blocked with TBS-T buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) containing 5% nonfat dry milk for 1 h at room temperature. Primary Antibody Dilution Buffer (Beyotime, P0023A-100ml) is used to dilute primary antibodies to working concentrations, then the membranes were incubated with primary antibodies at 4°C overnight. The HRP-con jugated goat antiMouse IgG secondary antibodies (Boster, Wuhan, China, BA1051) or HRP-conjugated goat antiRabbit IgG secondary antibodies (Boster, BA1055) were diluted in TBS-T (1:5,000 ratio). Next, the membranes were incubated with secondary antibod ies for 1 h at room temperature. The blots were developed using the BeyoECL Plus (Beyotime, P0018S) and imaged with an Amersham Imager 600 (GE Healthcare) imaging system.

Cellular fractionation
Cells were washed twice in ice-cold LS buffer (20 mM Hepes pH 7.8, 0.5 mM DTT, 0.5 mM MgCl 2 in water) and allowed to swell on ice for 20 min. 293T cells were gently scraped and disrupted on ice. The lysates were centrifuged at 6,006 g for 2 min at 4°C to pellet the nuclei. The supernatant (cytoplasmic fraction) was removed. The nuclei were washed five times in PBS, placed in nuclei resuspension buffer (50 mM Tris-HCl pH 8, 0.5 mM MgCl 2 , 20 mM iodoactetamide supplemented with protease inhibitor (Roche) and sonicated. Proteins were resolved by SDS-PAGE and detected by Western blot.

Promoter activity assay
293T cells were co-transfected with 100 ng of full length, a series of truncated or mutant promoter fireflyluciferase reporter constructs, and 10 ng of Renilla luciferase vector (pRL-TK). Luciferase activities were determined with the Dual Luciferase Reporter Assay System (Promega) and expressed as relative luciferase activity by normalizing firefly luciferase activity against Renilla luciferase activity, according to the manufacturer's protocol.

Chromatin immunoprecipitation (ChIP)
ChromaFlash High-Sensitivity ChIP kit (Epigentek, Farmingdale, NY, USA) was used to perform ChIP assay according to the manufacturer's protocol. Cells were infected with IAV (MOI = 0.01) for 36 h or not. The growth media of cells were removed, and cells were rinsed three times with cold PBS. Cells were added with formaldehyde to a final concentration of 1% and incubated at room temperature for 15 min. Glycine was added to cells to a final concentration of 125 mM to stop the cross-linking reaction and then sonicated to a fragment size range of 100-700 bp. Immunoprecipitation was performed by incubating sheared chromatin overnight at 4˚C with antiCREB antibody (CST, #9197) or rabbit IgG isotype (CST, #3900) and protein A + G Agarose beads (Santa Cruz Biotechnology, Cat# sc-2003). DNA precipitated from the samples was subjected to PCR amplification detecting a segment of miR-200b-3p promoter region using the primers: 5′-CCCAGGACCCAAAGCTGGTG-3′ (F) and 5′-AGTAAGATGGCCACGGCTGC-3′ (R). PCR products were resolved by 2% agarose gel electrophoresis and visualized using UV light. Input chromatin relative to its abundance was used to determine the expression level of a target DNA sequence.

Northern blot
Northern blot hybridization was performed by a nonradioactive method. Locked nucleic acid (LNA)modified probes were synthesized and 3′ end labeled with digoxigenin by Tsingke Biotech (Wuhan, China). RNAs of < 200 nucleotides were purified from cultured cells using MiPure Cell/Tissue miRNA Kit (Vazyme, RC201) following the manufacturer's instructions. Samples of 20 mg of total RNA were analyzed using a 15% polyacrylamide gel and transferred to Hybond-N + nylon membranes. Hybridization of membranes with digoxigenin-labeled DNA probes was performed as previously reported (35). Signal detection was performed as described in the manual for a DIG High Prime DNA labeling and detection starter kit II (Roche, Switzerland). The probes used for Northern blot hybridizations are listed here, U6: CGaATtTGcGTgTCaTCcTTgC and miR-200b-3p: TcaTCaTTaCCaGGcAGtATtA, the lowercase letters represent LNAmodified nucleotide.

Dual-luciferase reporter assays
Luciferase reporter vectors containing WT TBK1 3′ UTR or MUT TBK1 3′ UTR were co-transfected with the control mimic or miR-200b-3p mimic into HEK-293T cells to validate the miR-200b-3p targeting TBK1. The transfected cell lysates were analyzed by using the dual luciferase assay kit (Promega) at 24 h post-transfection. All obtained luciferase values were normalized against the Renilla luciferase control. HEK-293T cells grown in 48-well plates were co-transfected with luciferase reporter plasmids (IFN β-Luc, IRF3-Luc, ISRE-Luc, or NF-κB-Luc) and the pRL-TK plasmid to detect activation of the IFN pathway, along with the indicated amount of empty vector or miRNAs. 293T cells were left untreated or were treated with SeV for additional 12 h. The dual luciferase assay kit (Promega) was used to prepare and analyze cell lysates for firefly and Renilla luciferase activities.

Confocal microscopy
HEK-293T cells seeded on 14 mm coverslips were transfected with miRNAs or infected with SeV or IAV. 293T cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then stained with antibodies against CREB, IAV-NP, VSV-G, SeV-HN, IRF3, p65, or DAPI after incubation. 293T cells were incubated with Alexa 488-conjugated or 594-conjugated secondary antibodies for 1 h at room temperature after being washed three times. Staining was visualized with a ZEISS LSM 880 confocal microscope under an oil objective (Carl Zeiss AG, Oberkochen, Germany).

Mouse infection
The 6-week-old female C57BL/6 mice were randomly divided into indicated groups and infected intranasally (i.n.) with 200 plaque-forming units (PFU) of IAV or 1 × 10 7 FFU VSV or mock infected with DMEM in a volume of 20 µL. Changes in the mice's body weight and mortality were monitored daily. Mice that lost more than 25% of initial weight were humanely euthanized with CO 2 . The brains and lungs were collected for qPCR, histopathology, or immunohistochemistry analysis.

ELISA
ELISA was performed to quantify the amount of IFN-β in the 293T cell culture superna tants. Commercially available Human IFN-β ELISA kits (RayBiotech, Atlanta, GA, USA) were used following the manufacturer's instructions.

Histopathological analysis and immunohistochemistry (IHC)
PBS was used to perfuse the mice intracardially. Brains or lungs were removed and placed in 4% paraformaldehyde at room temperature for 12 h. After dehydration and wax immersion, samples were embedded in paraffin and sectioned into 4 µm. Sections were stained with hematoxylin and eosin (H&E) for histopathological analysis. Sections were processed using antigen retrieval and endogenous peroxidase quenching followed by anti-IAV-NP or anti-VSV-G antibody staining for the immunohistochemical analysis.

Statistical analysis
Data were expressed as the mean and standard deviation (SD). A student's t-test was performed to analyze the significant differences between the two groups. The log-rank (Mantel-Cox) test was used to analyze the survival ratio. The asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001). GraphPad Prism software, version 8.0 (GraphPad Software, La Jolla, CA, USA) was used to analyze and plot graphs.
Since miR-200b-3p displayed the most pronounced inhibitory effect on IFN-β expression among the miR-200 family, we then focused on miR-200b-3p in the following study. We used stem-loop qRT-PCR (stem-loop qPCR) assay to detect miR-200b-3p, which is highly accurate for miRNA detection, and only 0.1-3.7% nonspecific signal was observed for the miRNAs that differed by a single nucleotide (33,39,40). To further prove that our detection of miR-200b-3p is specific and does not detect any other miR-200 family members, we separately transfected cells with miR-200a/200b/200 c/141/429 mimics and then used primers targeting miR-200b-3p for stem-loop qPCR analysis. The results showed that the primers targeting miR-200b-3p could specifically detect miR-200b-3p but not other miR-200 family members (Fig. S1A).
We then examined miR-200b-3p levels in 293T cells infected with IAV or VSV at different time points by stem-loop qPCR to characterize miR-200b-3p expression upon viral infection. The results showed that mature miR-200b-3p was constitutively expressed in cells and increased significantly in a time-dependent manner after IAV or VSV infection ( Fig. 1C and 1D). Cells inoculated with UV-inactivated IAV or VSV showed no significant change in the expression level of miR-200b-3p ( Fig. 1C and D), suggesting that miR-200b-3p expression was triggered by viral replication. In addition, miR-200b-3p increased in a dose-dependent manner upon IAV or VSV infection at different MOIs ( Fig.  S1B and C). We also used the Northern blot assay and showed that IAV or VSV infection increased the expression of mature miR-200b-3p in a time-dependent manner ( Fig. S1D and G). We then measured the expression of the primary miR-200b-3p transcript (pri-miR-200b-3p) and the miR-200b-3p precursor (pre-miR-200b-3p) in virus-infected cells, from which the mature miR-200b-3p is processed. As expected, the expression of pri-miR-200b-3p and pre-miR-200b-3p showed a time-dependent increase in cells after IAV  nucleus (blue). Student's t-test was used for statistical analysis of comparisons between groups. Bar graph shows the means ± SD, n = 3. Scale bar = 10 µm.

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We then examined the effects of the classical inflammatory activator LPS on miR-200b-3p, as transcriptional regulatory effects of inflammatory pathways on microRNAs have been widely reported (16,41). To demonstrate the specificity of the probe for miR-200b-3p, we separately transfected miR-200 family members' mimics into cells and then detected by Fluorescence in situ hybridization (FISH). The results in Fig. S2A showed that the probes targeting miR-200b-3p could only specifically detect miR-200b-3p. FISH assay was further used and demonstrated that miR-200b expression was increased in IAV-infected or LPS-treated cells compared to mock-treated cells (Fig.  1E). Similarly, stimulation by LPS or VSV infection caused a significant increase of miR-200b-3p in 293T cells (Fig. 1F). Meanwhile, the application of asperuloside (42), a broad-spectrum inflammatory inhibitor, significantly downregulated IAV-or VSV-induced expression of in miR-200b-3p in cells ( Fig. 1E and F). Meantime, we used confocal microscopy to confirm IAV and VSV infection under the same experimental conditions ( Fig. S2B and C). Taken together, the above results suggested that IAV and VSV infection upregulated the miR-200b-3p expression through activation of inflammatory pathways.

IAV infection upregulates miR-200b-3p through ERK and p38 pathways
We then sought to determine the exact inflammatory pathway(s) responsible for the regulation of miR-200b-3p expression after confirming that inflammatory pathway activation was involved in the upregulation of miR-200b-3p. We found that IAV infection promoted the phosphorylation levels of p65, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK), and p38 in a time-dependent manner ( Fig. 2A), suggesting that IAV infection activated NF-κB and mitogen-activated protein kinase (MAPK) pathways, which is consistent with a previous report (43). The signal transduction pathways mediated by NF-κB and MAPK contributed to the activation of transcription factors (16,41). Transcription factors bind to miRNA regulatory elements and further regulate miRNA expression.
We then treated cells with LPS for the indicated time periods, and the R1 reporter activity of miR-200b-3p was confirmed to be upregulated (Fig. 2C), suggesting that the R1 reporter activity of miR-200b-3p was regulated by the activation of inflammatory signaling pathways. Specific inhibitors of these signaling pathways were then used to analyze which inflammatory signaling pathway plays an important role in the IAVinduced expression of miR-200b-3p. The results of the luciferase activity assay showed that IAV-induced miR-200b-3p promoter activity was significantly reduced in cells treated with p38specific or ERKspecific inhibitor compared to DMSO-treated cells (Fig. 2D). In contrast, cells treated with NF-κBspecific or JNKspecific inhibitors had no apparent inhibitory effect (Fig. 2D). Stem-loop qPCR analysis also showed that either the p38 specific inhibitor or the ERKspecific inhibitor significantly downregulated IAV-induced miR-200b-3p (Fig. 2E). Furthermore, stem-loop qPCR results showed that overexpression of p38 or ERK upregulated the expression of miR-200b-3p (Fig. S3). Taken together, these results indicate that IAV infection upregulates miR-200b-3p through the ERK and p38 signaling pathways.

CREB regulates miR-200b-3p promoter activity
To further clarify which transcription factor controls the expression of miR-200b-3p, CONSITE and JASPAR software were used for bioinformatics analyses to predict potential transcription factor-binding sites in the miR-200b-3p promoter (R1) (44,45). The putative binding sequences for two potential transcription factors, CREB and c-Fos, which are involved in the MAPK pathway (26), were found within the miR-200b-3p promoter region (R1). To determine whether the predicted transcription factor was responsive to the regulation of miR-200b-3p, a series of promoter constructs, including wild type (R1-wildtype), CREB binding site mutation (R1-mut-CREB) (TGTCCTCA to ACTGCAGT), and c-Fos binding site mutation (R1-mut-c-Fos) (CTGCCTCA to CACCAAGT), were generated and tested for their promoter activity under IAV infection. The results showed that mutation of the c-Fos binding site had no significant effect on constitutive or inducible lucifer ase activity (Fig. 3A). However, R1 with mutation of the CREB binding site showed significantly less response to IAV infection compared with the wild type, suggesting that the activation of miR-200b-3p promoter by IAV infection requires CREB (Fig. 3A). Furthermore, the overexpression of CREB significantly enhanced the promoter activity and the amount of miR-200b-3p ( Fig. 3B and 3D), whereas knockdown of CREB by using siRNAs had a suppressive effect ( Fig. 3C and 3D). Also, the CREBspecific inhibitor could inhibit the expression of miR-200b-3p in a dose-dependent manner (Fig. 3E).
Since phosphorylated transcription factors are transported to the nucleus and bind to the corresponding DNA fragments to exert their functions, we then tested whether IAV infection promoted the phosphorylation and nuclear translocation of CREB. Western blot results showed that IAV infection promoted CREB phosphorylation in a time-dependent manner (Fig. 3F). Consistently, Western blot results showed that the translocation of CREB from the cytoplasm to the nucleus increased upon IAV infection (Fig. 3G). In addition, confocal microscopy analysis showed that CREB translocated into the nucleus after IAV infection (Fig. 3H). Chromatin immunoprecipitation assay (ChIP) was then performed and confirmed that IAV infection could increase the binding of CREB to the miR-200b-3p promoter (Fig. 3I). Furthermore, the binding of CREB to the miR-200b-3p promoter was attenuated by ERK inhibitor and p38 inhibitor upon IAV infection, while the binding activity remained unchanged in cells treated with NF-κB inhibitor or JNK inhibitor (Fig.  3J). This result is consistent with CREB acting as a transcription factor downstream of the ERK and p38 pathways. In addition, Western blot results showed that UV-inactivated IAV and VSV (UV-IAV and UV-VSV) did not activate p38, ERK pathway, and downstream CREB ( Fig. S4A and B), which is consistent with the data that UV-IAV and UV-VSV did not upregulate miR-200b-3p expression. These results demonstrate that CREB plays an important role in IAV-or VSV-induced transcriptional regulation of miR-200b-3p.

MiR-200b-3p directly targets 3′ UTR of TBK1 mRNA
To investigate the potential mechanism of miR-200b-3p in the regulation of IFN-I production, we examined its specific target. Using TargetScan 7.0 and miRanda, miRNA target-prediction algorithms (46)(47)(48), the 3′ UTR of TBK1 mRNA showed a potential putative target site for miR-200b-3p. Since TBK1 is essential for the production of IFN-I, we focus on the targeting of miR-200b-3p to TBK1. We co-transfected cells with miR-200b-3p mimic and wild type TBK1 3′ UTR luciferase reporter plasmid and found that luciferase levels were significantly reduced. In contrast, application of miR-200b-3p inhibitor resulted in a significant increase in luciferase activity (Fig. 4A). Furthermore, a 4 bp mutation in the miR-200b-3p target sequence abolished the negative effect of miR-200b-3p on the expression of the TBK1 3′ UTR reporter construct (Fig. 4A). Also, to prove the targeting specificity of miR-200b-3p mimic and inhibitor, we transfected miR-200b-3p mimic and inhibitor into cells separately, and then measured the levels of miR-200b family members. The stem-loop qPCR results showed that cells transfected with miR-200b-3p mimic only significantly upregulated the amount of miR-200b-3p, We then performed an RNA immunoprecipitation (RIP) assay using Flag-tagged Ago2, which is a commercial component of the miRNAs-silencing complex (34), to confirm that TBK1 mRNA directly interacts with miR-200b-3p. The results showed that the TBK1 mRNA was significantly enriched in the miR-200b-3p group (Fig. 4B). Cells were co-transfected with miR-200b-3p mimics and TBK1 expression vector with WT or mutated 3′ UTR, and the expression of TBK1 in 293T cells was then measured. As expected, overexpression of miR-200b-3p reduced TBK1 levels in cells transfected with WT 3′ UTR TBK1, whereas TBK1 with mutated 3′ UTR was unaffected by miR-200b-3p (Fig. 4C). These results suggest that the nucleotide sequence in the TBK1 3′ UTR is a targeting site by miR-200b-3p.
We then transfected cells with pre-miR-200b-3p plasmid and infected them with SeV to confirm whether miR-200b-3p regulates endogenous TBK1. Western blot results showed that the pre-miR-200b-3p plasmid had a dose-dependent inhibitory effect on the protein level of TBK1 and the phosphorylation of IRF3, the downstream innate immune effector of TBK1, while the pre-miR-200b-3p plasmid had no effect on the expression of RIG-I and MAVS, which are the upstream effectors of TBK1 (Fig. 4D). Also, the pre-miR-200b-3p plasmid had a significant inhibitory effect both on the mRNA and protein levels of TBK1. Similarly, the applications of miR-200b-3p mimic suppressed TBK1 protein and mRNA levels, and the inhibition of TBK1 was restored in the presence of miR-200b-3p inhibitors (Fig. 4E). Interestingly, like most known and functionally well defined miRNA binding sites, the miR-200b-3p binding site within the TBK1 3′ UTR is highly conserved in vertebrates (Fig. 4F). Taken together, these data suggest that TBK1 mRNA is a direct target of miR-200b-3p and TBK1 expression is regulated by miR-200b-3p.

MiR-200b-3p negatively regulates IRF3 and NF-κB signaling pathway
During viral infection, IRF3 and NF-κB can be phosphorylated by the protein kinase TBK1 and subsequently translocated to the nucleus as activated transcription factors (9,49). Therefore, we investigated whether miR-200b-3p affects the IRF3 and NF-κB signaling pathways by targeting TBK1. The dual-luciferase reporter assays showed that miR-200b-3p mimics significantly impaired the activity of IRF3 and NF-κB, while the administration of miR-200b-3p inhibitor promoted the activity (Fig. 5A and B). We also tested and confirmed that transfection of pre-miR-200b-3p plasmid could downregulate the activation of IRF3 and NF-κB in a dose-dependent manner (Fig. S6A and B). The effect of miR-200b-3p on the nuclear translocation of endogenous IRF3 and NF-κB was then assessed by Western blot. MiR-200b-3p mimics transfection attenuated SeV-induced nuclear translocation of IRF3 and NF-κB p65 subunits (Fig. S6C and D). Consistently, we used confocal microscopy analysis and found reduced nuclear translocation of IRF3 and NF-κB p65 subunit in miR-200b-3p-overexpressed cells upon SeV infection (Fig. 5C and  D). Furthermore, Western blot results showed that miR-200b-3p also suppressed the phosphorylation of IRF3, IRF7, p65, and ikbα ( Fig. S6E and F). Correspondingly, treatment with miR-200b-3p inhibitor significantly increased the phosphorylation of IRF3, IRF7, p65, and ikbα in cells (Fig. S6E and F).
To further confirm that the regulation of IRF3 and p65 by miR-200b-3p was associated with TBK1, cells were co-transfected with miR-200b-3p mimics and TBK1 expression vectors with WT or mutated 3′ UTR, and the phosphorylation of IRF3 and p65 were determined. As shown in Fig. 5E and F, transfection with pre-miR-200b-3p plasmid had a dose-dependent inhibitory on the phosphorylation of IRF3 and p65. Consistently, the mutant 3′ UTR of TBK1 rescued the inhibition of phosphorylation (Fig. 5G and H). Taken

MiR-200b-3p inhibits TBK1-mediated type I interferon production
Previous studies have shown that TBK1 can activate both NF-κB and IRF3 signaling to induce IFN-I expression (3)(4)(5). IFN-I then leads to the activation of the JAK-STAT signaling cascade and induces the expression of many ISGs (50). Therefore, we investigated whether miR-200b-3p inhibits IFN-I production by targeting TBK1. The results of dual luciferase reporter assays showed that miR-200b-3p mimics could reduce IFN-β and ISRE reporter activity, while the inhibitors of miR-200b-3p improved the activity (Fig. 6A). We also observed that transfection with pre-miR-200b-3p expression plasmid inhibited IFN-β and ISRE reporter activity in a dose-dependent manner (Fig. S7A and B). Next, we investigated whether the nuclear translocation and phosphorylation of STAT1 were regulated by miR-200b-3p. We found that SeV-induced nuclear translocation of STAT1 was inhibited by miR-200b-3p (Fig. 6B). Consistently, miR-200b-3p mimics attenuated phosphorylation of STAT1, while miR-200b-3p inhibitors increased the level of phosphorylation (Fig. 6C). Furthermore, the effects of miR-200b-3p on SeV-stimulated expression of ISG15, which is one of the most rapidly and strongly induced ISGs (51,52), was evaluated by Western blot. As expected, miR-200b-3p expression significantly suppressed ISG15 protein levels, and the suppression of ISG15 could be reversed in the presence of miR-200b-3p inhibitors (Fig. 6C).
We then co-transfected pre-miR-200b-3p expression plasmid and TBK1 expression plasmid with WT or MUT 3′ UTR to further confirm that the regulation of ISG15 by miR-200b-3p was associated with TBK1. The results showed that pre-miR-200b-3p has a dose-dependent inhibitory effect on the protein levels of ISG15 in the presence of TBK1 with WT 3′ UTR (Fig. 6D). However, there is no change in the ISG15 protein level was observed when co-transfected with TBK1 with MUT 3′ UTR (Fig. 6E). To further investigate the function of miR-200b-3p in IFN response, SeV was used as a stimulus to treat cells, and then the protein level of IFN-β was measured by ELISA. Inhibition of miR-200b-3p significantly increased IFN-β production, whereas expression of miR-200b-3p inhibited IFN-β production (Fig. 6F). Taken together, these results demonstrate that miR-200b-3p negatively regulates IFN-I production.

Inhibition of miR-200b-3p restricts IAV replication in cells and attenuates viral pathogenicity in mice
Prevention and treatment of IAV infection are challenging due to its high mutational potential, numerous subtypes, and wide host range (53). As a key component of the innate immune system, rapid and robust induction of IFN-I plays a critical role in host defense against IAV infection (54). Since the enhancement of interferon by miR-200b-3p silencing has been demonstrated, we further evaluated the effect of miR-200b-3p inhibition on IAV replication and pathogenicity. The results showed that miR-200b-3p increased the level of IAV NP protein, whereas the miR-200b-3p inhibition decreased the NP protein expression (Fig. 7A). Furthermore, we observed that overexpression of pre-miR-200b-3p promoted NP protein level in a dose-dependent manner (Fig. 7B). In addition, we co-transfected pre-miR-200b-3p and TBK1 with WT or MUT 3′ UTR and then infected cells with IAV to confirm that the promotion of IAV replication by miR-200b-3p was related to its targeting effect on TBK1. The results showed that pre-miR-200b-3p increased NP protein expression in cells transfected with TBK1 carrying the WT 3′ UTR (Fig. 7C), and there was no significant effect on cells transfected with TBK1 carrying the MUT 3′ UTR (Fig. 7D). The IAV titers in the supernatant of cells transfected with miR-200b-3p mimics or miR-200b-3p inhibitors at each time point were then measured by virus titration, which showed that miR-200b-3p mimics significantly promoted IAV replication. Consistently, miR-200b-3p inhibitors had an inhibitory effect on replication (Fig. 7E). These results suggest that miR-200b-3p may promote IAV replication by inhibiting IFN-I production through the degradation of TBK1.
The therapeutic potential of miR-200b-3p inhibition was then evaluated in an IAVinfected mouse model. We injected miR-200b-3p inhibitor and NC inhibitor, respectively, into mice after infection with lethal doses of IAV and monitored animal survival ratio. The results showed that miR-200b-3p inhibition significantly attenuated virus pathogenicity.  was analyzed by two-way ANOVA test. The log-rank (Mantel-Cox) test was used to analyze the survival ratio. Student's t-test was used for statistical analysis of comparisons between groups. Bar graph shows the means ± SD, n = 3. Western blot data are representative of at least three independent experiments.

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Injections of miR-200b-3p inhibitor after viral infection resulted in mild weight loss and 20% mortality. However, mice injected with NC inhibitor after viral infection showed severe disease and 80% mortality ( Fig. 7F and G). Notably, viral titers in the lungs of the IAV+NC inhibitor group at 3 dpi were more than 30-fold higher than those detected in the IAV+miR-200b-3p inhibitor group (Fig. 7H). Meanwhile, weaker NP antigen signals were observed in the IAV+miR-200b-3p inhibitor group than in the IAV+NC inhibitor group (Fig. 7I). Also, the lungs of the IAV+NC-inhibitor group had moderate to severe bronchiolar necrosis and pulmonary edema, whereas these pathological changes were rarely observed in the IAV+miR-200b-3p inhibitor group (Fig. 7I). These results demon strated that miR-200b-3p inhibition exerted a therapeutic role in IAV-infected mice. Taken together, we found that the CREB-mediated miR-200b-3p inhibits TBK1 and suppresses IFN-I production, through which the virus evades host's innate immune response. Inhibition of miR-200b-3p can block this signaling cascade and restrict viral replication (Fig. 7J).
We also evaluated miR-200b-3p inhibitors in vitro and in vivo by using another well-known RNA virus, VSV, which is interferon-sensitive (39), to further confirm that miR-200b-3p inhibitors restrict viral replication by regulating interferon production. In vitro, the miR-200b-3p inhibitor significantly reduced VSV-G expression and virus titers, whereas miR-200b-3p mimics increased both of them ( Fig. S8A and B). We also observed that pre-miR-200b-3p promoted VSV-G protein expression in a dose-dependent manner (Fig. S8C). In vivo, to silence endogenous miR-200b-3p, the chemically modified antisense oligonucleotide specific for miR-200b-3p was delivered to VSV-infected mice. All mice in the NC inhibitor group survived, while the mice in the VSV+NC inhibitor group had a lethality rate of 70%. In contrast, the VSV+miR-200b-3p inhibitor group showed a 60% reduction in lethality (Fig. S8D). In addition, the decrease in body weight was more pronounced in the VSV+NC inhibitor mice (Fig. S8E).
We also found that the VSV+NC inhibitor group contained higher viral titers than the VSV+miR-200b-3p inhibitor group (Fig. S8F). More VSV-G positive cells were consis tently observed in the VSV+NC inhibitor group (Fig. S8G). Hematoxylin and eosin staining was then performed, which showed that vascular engorgement and hyperplasia were observed in brains from the VSV+NC inhibitor group. In contrast, no significant pathological lesions were observed in mouse brains from the VSV+miR-200b-3p inhibitor group (Fig. S8). In addition, the mRNA levels of IFN and ISGs, including IFN-β, Mx1, OAS1a, IFIT3, and ISG15, in mouse brains were also increased in the VSV+miR-200b-3p inhibitor group (Fig. S8G). Collectively, these data indicate that miR-200b-3p affects VSV pathogenicity in vivo, including viral replication level and mouse survivor ratio.

MiR-200b-3p inhibitors suppress the replication of multiple pathogenic viruses
Viral infection of mammals induces the synthesis of type I IFN, which inhibits viral replication. The high susceptibility of type I IFN receptor knockout mice to infection by a variety of viruses provides strong evidence for the important role of the IFN system in protection against viral infection (55). We evaluated the effects of miR-200b-3p inhibitors on RABV (negative-stranded RNA virus), JEV (positive-stranded RNA virus), and HSV-1 (DNA virus), all of which pose a significant threat to public health worldwide.
After transfection with miR-200b-3p mimics or miR-200b-3p inhibitor for 24 h, N2a cells were respectively infected with different viruses, and then the expression of different viral proteins was examined separately. The results showed that miR-200b-3p increased the levels of RABV N protein (Fig. 8A), JEV E protein (Fig. 8B), and GFP (recombinant HSV-1 carrying the GFP gene) (Fig. 8C). Consistently, miR-200b-3p inhibition decreased the expression of these proteins. In addition, we observed that pre-miR-200b-3p promoted the expression of these proteins in a dose-dependent manner (Fig. 8D to 8F). After transfection with miR-200b-3p mimics or miR-200b-3p inhibitors for 24 h, N2a cells were infected with RABV, JEV, and HSV-1, respectively, and the viral titers of cell supernatants were measured at different time points. The results showed that miR-200b-3p mimics significantly promoted viral replication, whereas miR-200b-3p inhibitors had an inhibitory effect on viral replication (Fig. 8G through I). Taken together, these results indicate that miR-200b-3p inhibitor can be used as a broad-spectrum inhibitor against viral infection.

DISCUSSION
In this study, we identified miR-200b-3p as an IFN-I-related miRNAs, which inhibits the expression of TBK1 by directly binding with TBK1 3′ UTR. Upon viral infection, the upregulation of miR-200b-3p led to the reduction of TBK1-mediated IFN-I production and impaired the antiviral innate response. Importantly, miR-200b-3p inhibition has a broad-spectrum effect against viral replication by promoting IFN-I production both in vitro and in vivo. In addition, we found that activation of the transcription factor CREB by viral infection promotes the expression of miR-200b-3p. These data provided mechanis tic insight into how miRNAs affect virus survival and immune escape.
Currently, the most effective ways to protect people from viral infections are vaccina tion and the use of antiviral drugs. However, viruses that pose an ongoing threat to humans, such as IAV, have the ability to frequently outcompete vaccines due to rapid evolution and the emergence of variants that are resistant to available antiviral drugs. Therefore, there remains an urgent need to develop effective treatments with broadspectrum activity (56)(57)(58). IFN-I is effective against different groups of viruses through a variety of mechanisms (54). In addition, viruses can evade IFN inhibition by interfering with or disrupting molecules involved in IFN signaling (59). Here we identified a novel host factor involved in interferon regulation, miR-200b-3p.
Several studies showed that miRNAs regulate genes related to innate immune responses following viral infection. Previous studies suggested that enterovirus-induced miR-146a facilitates viral pathogenesis by suppressing IFN production by targeting IRAK1 and TRAF6 (60). Here, we further demonstrated that miR-200b-3p targets TBK1, through which miR-200b-3p inhibits TBK1-mediated NF-κB and IRF3 signaling. Interestingly, miR-429, which was found to inhibit the expression of IFN-β (Fig. 1B), also downregulates the TBK1 mRNA level (Fig. S9A). Since miR-429 also contains fragments complementary to the conservative sequence of TBK1 (CAGUAUU), it is possible that miR-429 inhibits the production of IFN-β by downregulating the level of TBK1, although the detailed mecha nism needs further investigation.
The miR-200-3p mimics negatively affect the production of IFN-I and ISG, whereas the inhibition of miR-200b-3p promotes the production. The use of miR-200b-3p inhibitor significantly inhibited the proliferation of several viruses in vitro, including HSV-1, VSV, IAV, JEV, and RABV, and the introduction of mutations in the binding site of TBK1 3′ UTR with miR-200b-3p could restore this inhibition. MiRNAs are thought to target multiple mRNAs, called the targetome, to regulate gene expression. A single miRNA can regulate the protein synthesis of thousands of genes through direct or indirect effects (61). We may be far from discovering the last target of miR-200b-3p, and some potential targets may also be involved in regulating antiviral responses, which may trigger interesting future work.
Inhibition of microRNA is a very promising discovery in terms of reducing viral load in vivo and suggests a new therapeutic strategy to control viral infection (60). miR-122 inhibition by a specific inhibitor has been shown to restrict HCV infection and replication in chimpanzee models and in phase II clinical trials (62,63). Thus, we introduced the miR-200b-3p inhibitor to demonstrate its potential application for viral treatment in the mouse model of IAV infection. Therapeutic administration at an early stage of viral infection effectively protected IAV-infected mice from virulent challenge. As previously described, this chemically modified miRNA inhibitor could cross the blood-brain barrier into the central nervous system via the intravenous route (64). The use of miR-200b-3p inhibitor significantly reduced mortality in a mouse model of VSV encephalitis, consistent with the important role of IFN in VSV pathogenecity. We further verified the relationship between miR-200b-3p, TBK1 and IFN production in vivo. In addition, the data provided new insights into how microRNA promotes viral survival and immune escape.
There is evidence that virus-induced host transcription factors induce miRNA expression by binding to their promoter regions (65). For example, the NF-κB p65 subunit binds to the promoter element of a subset of miRNAs genes and transcriptionally regulates their expression in response to LPS stimulation (66). In addition, miR-100, -146 a, and -150 were reported to be novel p53 and NF-κB p65/RelA responsive miRNAs (67). As a transcriptional regulator, CREB generally enhances the expression of target genes. Studies have shown that some noncoding CREB targets have been identified. CREB can directly bind to the regulatory sequences of miR-23a and increase miR-23a expression (68). CREB increases the expression of miR-373 by regulating its promoter (69). Here, we found that the promoter for miR-200b-3p contains a CREB binding site and that miR-200b-3p expression after IAV infection depends on CREB activation. NF-κB and MAPK pathway was activated upon IAV infection and played an important role in IAV-induced pathogenesis (70). Signal transduction pathways mediated by the MAPK family, including ERK1/2, JNK, and p38, contribute to the activation of transcription factors (71). Furthermore, ERK and p38 are potential upstream regulators in signaling CREB (72,73), and CREB is also found to be activated in IAV infection (74). The tran scription factors of microRNAs and their binding sites may differ or act synergistically under different stimulating factors, so the regulatory molecules of miR-200b-3p still need further investigation (20,60,75). In addition to the two RNA viruses, IAV and VSV, the DNA virus HSV-1 was also found to upregulate miR-200b-3p (Fig. S9B). Whether different types of viruses have the ability to regulate miR-200b-3p expression needs further research. In conclusion, we have identified a novel miRNA-innate immunity interaction in which CREB-mediated miR-200b-3p leads to the degradation of TBK1 mRNA and suppression of IFN production, thereby allowing the virus to evade host immune attack. Inhibition of miR-200b-3p could block the IFN signaling cascade, thereby alleviating symptoms and mortality. Our findings highlight that miR-200b-3p is a potential drug target for a wide range of viral infections.

ACKNOWLEDGMENTS
This study was supported by the National Key Research and Development Program of China (No. 2022YFD1800100). The funders had no conflict of interest in study design, data collection and analysis, publication decision, or manuscript preparation.