miR-193b represses influenza A virus infection by inhibiting Wnt/β-catenin signaling

Summary

Due to an increasing emergence of new and drug-resistant strains of the influenza A virus (IAV), developing novel measures to combat influenza is necessary. We have previously shown that inhibiting Wnt/β-catenin pathway reduces IAV infection. In this study, we aimed to identify antiviral human microRNAs (miRNAs) that target the Wnt/β-catenin signaling pathway. Using a miRNA expression library, we identified 85 miRNAs that upregulated and 20 miRNAs that downregulated the Wnt/β-catenin signaling pathway. Fifteen miRNAs were validated to upregulate and 5 miRNAs to downregulate the pathway. Overexpression of 4 selected miRNAs (miR-193b, miR-548f-1, miR-1-1 and miR-509-1) that downregulated the Wnt/β-catenin signaling pathway reduced viral mRNA, protein levels in A/PR/8/34-infected HEK293 cells and progeny virus production. Overexpression of miR-193b in lung epithelial A549 cells also resulted in decreases of A/PR/8/34 infection. Furthermore, miR-193b inhibited the replication of various strains, including H1N1 (A/PR/8/34, A/WSN/33, A/Oklahoma/3052/09) and H3N2 (A/Oklahoma/309/2006), as determined by a viral reporter luciferase assay. Further studies revealed that β-catenin was a target of miR-193b and β-catenin rescued miR-193b-mediated suppression of IAV infection. miR-193b induced G0/G1 cell cycle arrest and delayed vRNP nuclear import. Finally, adenovirus-mediated gene transfer of miR-193b to the lung reduced viral load in mice challenged by a sublethal dose of A/PR/8/34. Collectively, our findings suggest that miR-193b represses IAV infection by inhibiting Wnt/β-catenin signaling.

Introduction

Influenza A virus (IAV) infection causes annual epidemics and recurring pandemics, leading to substantial morbidity and mortality, as well as significant economic losses. IAV belongs to the family Orthomyxoviridae and has a segmented, negative-sense, and single-stranded RNA genome. Although vaccines remain a major means of prevention, a significant amount of time is required to develop and produce an effective vaccine against a new virus strain (Soema, Kompier, Amorij, & Kersten, 2015). Furthermore, vaccines need to be reformulated annually due to the frequent emergence of new viruses (Houser & Subbarao, 2015). Antiviral drugs, on the other hand, are essential for treatment as well as prophylaxis. However, the error-prone nature of the influenza RNA polymerase, due to its lack of proofreading-repair activity, makes the virus highly susceptible to mutation, resulting in its resistance to antivirals (Watanabe et al., 2014). For example, there has been rapid emergence of IAV strains that are resistant to amantadine and rimantadine, and these antivirals are thus no longer recommended for anti-influenza treatment (Barr et al., 2007; Bright et al., 2005). Resistance against neuraminidase inhibitors, such as oseltamivir and zanamivir as well as newly developed peramivir and laninamivir, has also been reported (Barrett & McKimm-Breschkin, 2014; Hurt et al., 2009; Kamali & Holodniy, 2013; Orozovic, Orozovic, Jarhult, & Olsen, 2014). Therefore, it is increasingly urgent to develop drugs that target host factors rather than viral proteins, which is less likely to cause drug resistance.

The small coding capacity of IAV requires it to utilize the host cell machinery for its life cycle (Watanabe, Watanabe, & Kawaoka, 2010; York, Hutchinson, & Fodor, 2014). Many host proteins and signaling pathways regulate IAV infection at different stages. Early in 2003, Wurzer et al. discovered that efficient IAV propagation depends on the activation of host caspase-3, a central player in apoptosis, as the presence of a caspase-3 inhibitor in cells strongly impairs viral replication (Wurzer et al., 2003). Several studies have shown that IAV stabilizes the p53 protein, activates p53 signaling and consequently induces apoptosis in host cells (Nailwal, Sharma, Mayank, & Lai, 2015; Turpin et al., 2005; Zhirnov & Klenk, 2007). Recently, cyclophilin A was found to interact with the IAV M1 protein and thus to impair early viral replication (X. Liu et al., 2012). IAV also interacts with many other cellular pathways, including the NF-κB, PI3K/Akt, MAPK, PKC/PKR and TLR/RIG-I signaling cascades, to overcome host defenses against the virus (Gaur, Munjhal, & Lai, 2011; Ludwig & Planz, 2008; C. Zhang et al., 2014).

MicroRNAs (miRNAs) are ~22-nt small non-coding RNAs that post-transcriptionally regulate gene expression by binding the 3’-untranslated region (3’-UTR) of a target mRNA to inhibit protein translation or degrade mRNA (Y. Wang, Strieker, Gou, & Liu, 2007). Several thousand miRNAs have been identified in plants, animals and viral genomes (Akhtar, Micolucci, Islam, Olivieri, & Procopio, 2016). miRNAs are key modulators in diverse signaling pathways (Zamore & Haley, 2005). Increasing evidence indicates that miRNAs also participate in host-virus interactions and play a pivotal role in the regulation of viral replication. For example, miR-122, a liver-specific miRNA, facilitates viral replication by targeting the 5’-UTR of hepatitis C virus (HCV) RNA (Thibault et al., 2015). Cellular miR-24 and miR-93 target the viral large protein (L protein) and phosphoprotein (P protein) genes of vesicular stomatitis virus (VSV) (Otsuka et al., 2007). In addition, miR-323, miR-491, and miR-654 inhibit replication of H1N1 influenza A virus by binding the PB1 gene (Song, Liu, Gao, Jiang, & Huang, 2010). miRNA can also modulate the type I interferon (IFN) system to combat viral infection. Wang et al. has demonstrated that IFN-induced miR-155 positively regulates the host antiviral innate immune response via type I IFN signaling by targeting suppressor of cytokine signaling 1 (SOCS1) (P. Wang et al., 2010).

We have recently demonstrated a beneficial role of Wnt/β-catenin signaling in IAV infection. The activation of Wnt/β-catenin with Wnt3a enhances influenza virus replication, whereas the inhibition of the pathway with iCRT14 decreases influenza infection (More et al., 2018). In the present study, we sought to identify miRNAs that regulate IAV replication by modulating the Wnt/β-catenin pathway. Here, we report the systematic screening of a miRNA expression library and identification of 20 miRNAs that modulate the activity of the Wnt/β-catenin pathway. Four selected miRNAs that inhibit the pathway, miR-193b, miR-548f-1, miR-1-1 and miR-509-1, were found to have anti-IAV activities. miR-193b suppressed IAV replication by targeting β-catenin.

Results

Screening of miRNAs that alter Wnt/β-catenin signaling activity

The Wnt/β-catenin signaling pathway plays an important role in various biological processes. Several studies have shown that a number of miRNAs modulate the activity of Wnt/β-catenin signaling by regulating the expression of the pathway-related components (Anton, Chatterjee, Simundza, Cowin, & Dasgupta, 2011; T. Hu et al., 2016; K. Huang et al., 2010). To systematically identify miRNAs that regulate Wnt/β-catenin signaling, we constructed a miRNA expression library composed of 738 human miRNA precursor expression vectors and performed screening using a TOPflash reporter luciferase assay as a readout for Wnt/β-catenin signaling activity (Fig. 1A). The TOPflash reporter vector contains three copies of TCF/LEF binding sites upstream of the luciferase reporter gene. The initial screening was performed in 96-well plates with 3 replicates. Human HEK293T cells were transfected with a TOPflash luciferase reporter plasmid, miRNA expression plasmid, and the normalization vector, pRL-TK vector for 24 h and were then stimulated with Wnt3a for 24 h, followed by the dual luciferase assay. Among 738 miRNAs, eighty-five miRNAs were found to upregulate and 20 to downregulate luciferase activity with a fold change ≥2, resulting in a hit rate of 14.2% (Fig. 1A and Table S1).

Fig. 1. Screening of miRNAs regulating Wnt/β-catenin signaling.

(A) Schematic of miRNA screening. (B) Summary of miRNA hits in Wnt/β-catenin signaling screening. (C-F) miRNA hit verification. HEK293 cells were transfected with a miRNA plasmid or its control vector (miR-Con) together with the TOPflash (wild-type TCF binding sites) or FOPflash (mutated TCF binding sites) luciferase reporter vector and were stimulated with 50% Wnt3a-conditioned medium. TOPFlash or FOPlash firefly luciferase activity was normalized to pRL-TK Renilla luciferase activity. The results are expressed as the ratio of miRNA/miR-Con (C, D) and ratio of TOPflash/FOPflash (E, F). A TOP/FOPflash ratio of ≥ 1.5 was used as a cut-off value.

To further validate the primary screen data, we repeated the reporter assay with a FOPflash vector containing mutated TCF binding sites as a negative control for TOPflash activity. If a miRNA changed the FOPflash activity similar to the TOPflash activity, the miRNA was considered to be a false positive. We selected approximately equal numbers of up- and down-regulating miRNAs (up, 24 miRNAs with a ≥ 3-fold change, and down, 20 miRNAs with a ≥2-fold change, Fig. 1B). Among the 20 down-regulating miRNAs, miR-548f-1, 548f-2 and 548f-4 have an identical mature miRNA and thus miR-548f-2 and 548f-4 were excluded. TOPFlash and FOPlash firefly luciferase activities, normalized to pRL-TK Renilla luciferase activity are shown in Fig. 1C for the up-regulating miRNAs and Fig. 1D for down-regulating miRNAs. A ratio of normalized TOPflash/normalized FOPflash are presented in Fig. 1E, F. Using a TOPflash/FOPflash ratio of 1.5 as a cut-off value, we identified 15 and 5 miRNAs that up- and downregulated the Wnt/β-catenin signaling pathway, respectively.

Identification of miRNAs with anti-IAV activities

We then tested whether the above-identified miRNAs that downregulated the Wnt/β-catenin pathway could inhibit IAV infection. Among the 5 miRNAs (miR-193a, miR-193b, miR-548f-1, miR-1-1 and miR-509-1) that downregulated Wnt/β-catenin signaling, miR-193a and miR-193b are isoforms of the same family. Because miR-193b showed greater inhibition of Wnt/β-catenin signaling than miR-193a, we tested the anti-IAV activities of miR-193b and the other miRNAs. We overexpressed each miRNA in HEK293 cells for 24 h, infected the cells with A/PR/8/34 at an MOI of 0.01 for 48 h and determined viral mRNA and protein expression in infected cells as well as the titer in the culture medium. The miRNA expression vector contains an EGFP marker, which can be used to monitor the transfection efficiency. EGFP images showed an approximately 75-85% transfection efficiency (Fig. 2A), and overexpression of miRNAs was confirmed by real-time PCR (Fig. 2B). Western blot analysis showed that overexpression of miR-193b, miR-548f-1, miR-1-1 and miR-509-1 in HEK293 cells reduced viral NP protein expression by 73 ± 14%, 42 ± 9%, 61 ± 25%, and 35 ± 19%, respectively, and NS1 protein expression by 98 ± 1%, 61 ± 21, 63 ± 18, and 30 ± 15%, respectively (Fig. 2C, D). These miRNAs also reduced the mRNA levels of NP, NS1 and PB1 in cells by 30-60% (Fig. 2E). Furthermore, progeny virus production in the culture media was suppressed by all of the miRNAs (Fig. 2F). Immunostaining of NP revealed that these miRNAs also led to a decrease in IAV-infected cells (Fig. 2G). It is noteworthy that miR-509-1 inhibited the Wnt/b-catenin signaling the second most but merely showed moderate suppression of the IAV infection, suggesting a possible off-target effect of the miRNA. Taken together, the data showed that the miRNAs that downregulated the Wnt/β-catenin signaling pathway reduced IAV infection.

Fig. 2. Effects of miRNAs that upregulate Wnt/β-catenin signaling on IAV infection.

(A, B) HEK293 cells were transfected with a pENTR-miRNA or its control vector (miR-Con) for 72 h. The transfection efficiency was monitored by the GFP signal and the cells were counter-stained with Hoechst 33342 (blue). miRNA expression was determined by real-time PCR, normalized to β-actin and expressed as a ratio to miR-Con. (C-F) HEK293 cells were transfected with pENTR-miRNA or miR-con for 24 h, followed by infection with A/PR/8/34 at an MOI of 0.01 for 48 h. NP and NS1 protein levels were determined by western blotting, densitometrically quantified and normalized to β-actin. NP, NS1 and PB1 mRNA expression was determined by real-time PCR and normalized to β-actin. Virus yields in the medium were titrated by the TCID₅₀ assay. (G) HEK293 cells were transfected with a pENTR-miRNA or miR-con vector for 24 h, followed by infection with PR8 IAV at an MOI of 0.5 for 16 h. Immunofluorescent staining was performed to detect viral NP (red). GFP images show cells overexpressing the miRNAs. The cells were counter-stained with Hoechst 33342 (blue). Data are expressed as the mean ± SE of three independent experiments. *P<0.05 vs. miR-Con, **P<0.01 vs. miR-Con*** P<0.001 vs. miR-Con and ****P<0.0001 vs. miR-Con (B and F: One-way ANOVA, Bonferroni’s multiple comparisons test; D and E: Two-way ANOVA, Bonferroni’s multiple comparisons test).

Further characterization of the anti-IAV activities of miR-193b

Given that miR-193b suppressed Wnt/β-catenin signaling and the viral mRNA and protein levels more than other miRNAs, we selected miR-193b for further investigation. First, we determined whether the effects of miR-193b on IAV infection were cell type-dependent. The lung is the primary site of IAV infection, and thus, we chose human lung epithelial A549 cells for this purpose. Due to the low transfection efficiency of plasmid into A549 cells, we used a lentivirus to overexpress miR-193b. Lentivirus infection efficiencies under our conditions were nearly 100% as revealed by EGFP images (Fig. 3A) although the intensities of GPF varied among cells. The miR-193b level, as determined by real-time PCR, was increased 26-fold over miR-Con in A549 cells (Fig. 3B). Cell viability was not affected by lentivirus infection as determined by CellTiter Blue assay (Fig. 3C). Overexpression of miR-193b reduced the viral NP and NS1 protein levels in both multiple cycle (Fig. 3D, E) and single cycle (Fig. 3H, I) of infection. Overexpression of miR-193b also decreased the mRNA levels of NP, NS1 and PB1 by 30% to 43% (Fig. 3F) and progeny virus production in the culture medium by more than one log (Fig. 3G). These results indicated that the anti-IAV activity of miR-193b was not cell type-dependent.

Fig. 3. Effects of miR-193b on IAV replication in A549 cells.

(A, B) Verification of miRNA overexpression. A549 cells were infected with the miR-193b or miR-Con lentivirus at an MOI of 100 for 48 h. The infection efficiency was monitored by the GFP signal, and the cells were counter-stained with Hoechst 33342 (blue). miR-193b expression was determined by real-time PCR and normalized to miR-Con. (C) Cell viability at 72 h after lentivirus infection determined by a CellTiter Blue assay and normalized to non-infected cells (blank). (D-G) Effects of miR-193b on IAV infection. A549 cells were infected with miR-193b or miR-Con lentivirus (MOI 100) for 48 h, followed by infection with A/PR/8/34 (MOI 0.01) for 48 h. NP and NS1 protein expression was detected by western blotting, densitometrically quantified and normalized to β-actin. Viral NP, NS1 and PB1 mRNA expression was determined by real-time PCR and normalized to β-actin. The virus yield in the medium was titrated by the TCID₅₀ assay. (H-I) miR-193b or miR-Con lentivirus-infected A549 cells were infected with IAV at MOI 0.5 for 8 h. NP and NS1 protein levels in the infected cells were detected by western blotting and quantified. Data are expressed as the mean ± SE of three independent experiments. *P<0.05 vs. miR-Con and **P<0.01 vs. miR-Con (B and G: Student’s t-test; C: One-way ANOVA, Bonferroni’s multiple comparisons test; E, F and I: Two-way ANOVA, Bonferroni’s multiple comparisons test).

We then assessed whether the effects of miR-193b on IAV infection were dependent on the viral strain using an IAV luciferase reporter assay (More et al., 2018). The IAV luciferase reporter vector contains a firefly luciferase under control of NP 5’ and 3’ UTRs of influenza A/WSN/33 so it is able to respond to IAV. Luciferase reporter activity was detected after reconstitution of the IAV polymerase complex after virus infection. The reporter vector was transfected into HEK293 or A549 cells along with the miR-193b plasmid or miR-con vector. The cells were then infected with A/PR/8/34, A/WSN/33, OK/09 H1N1 and A/Oklahoma/309/2006 H3N2 at the indicated MOIs for 48 h, followed by the dual luciferase assay. The viruses were chosen because A/PR/8/34 and A/WSN/33 are the most representative laboratory H1N1 strains, while OK/09 is a pandemic H1N1 virus and H3N2 is a virus strain that causes seasonal flu annually as well as endemic outbreaks. To obtain a proper readout of the luciferase activities, we first optimized the dose of the viruses with MOIs of 0.001, 0.005, 0.01, 0.05 and 0.1. The dose that resulted in a ratio (firefly/renilla) between 10 and 100 was selected (data not shown). The results showed that overexpression of miR-193b inhibited IAV infection of all tested strains by 45-73% in HEK293 cells and by 25-76% in A549 cells (Fig. 4A, B), indicating that the anti-viral effect of miR-193b was not strain-dependent.

Fig. 4. Effects of miR-193b on different strains of IAV replication.

HEK293 or A549 cells were transfected with a pENTR-miR-193b or control vector (100 ng), the IAV firefly luciferase reporter plasmid vNP-luc/pHH21 (20 ng) and pRL-TK Renilla plasmid (5 ng) for 24 h. The cells were then infected with different strains of IAV, A/PR/8/34, OK/09, A/WSN/33 and A/Wisconsin/67/05 at an MOI of 0.01, 0.05, 0.005 and 0.01, respectively, for 48 h. Firefly luciferase activity was normalized to pRL-TK Renilla luciferase activity in HEK293 (A) and A549 (B) cells. The results are expressed as a ratio to miR-Con-transfected cells. Data shown are the mean ± SE of three independent experiments. *P<0.05 vs. miR-Con, **P<0.01 vs. miR-Con and ***P<0.001 vs. miR-Con (Two-way ANOVA, Bonferroni’s multiple comparisons test).

Knockout of miR-193b with CRISPR/Cas9 enhances IAV infection

To determine the effects of endogenous miR-193b on IAV infection, we knocked out miR-193b with CRISPR/Cas9 and determined their effects on IAV infection. Two sgRNAs were designed to target two regions of pre-miR-193b (Fig. 5A). A549 cells were infected with a sgRNA-lentiCRISPR virus or a control virus, and one clone was chosen from each sgRNA after puromycin selection. The surveyor mutation assay showed that the mutations were generated in both miR-193b knocked-out clones (Fig. 5B). DNA sequencing revealed that one base was inserted in the sgRNA1 clone and two bases were deleted in the sgRNA2 clone (Fig. 5C). miR-193b expression was significantly reduced with both clones (Fig. 5D), likely due to the disruption of miRNA processing (Chang et al., 2016). When miR-193b-knocked out A549 cells were infected with A/PR/8/34 at an MOI of 0.01 for 48 h, viral NP protein level was significantly increased in both clones (Fig. 5E, F). Progeny virus production in the culture medium was increased in the knock-out cells, but only reaching a significant level in the sgRNA2 clone (Fig. 5G). The results further support that miR-193b negatively regulates IAV infection.

Fig. 5. Effect of miR-193b knockout on IAV infection.

(A) Target regions of two sgRNAs. (B) CRISPR/Cas9-mediated mutation as detected by the surveyor mutation assay. (C) DNA sequencing showing insertion (green) and deletions (red) generated by CRISPR/Cas9 in the miR-193b knocked out clones. (D) miR-193b expression in the miR-193b knocked out clones by CRISPR/Cas9 as determined by real-time PCR. (E-G) sgRNA1, sgRNA2 or control vector (Con)-treated A549 cells were infected with A/PR/8/34 at MOI 0.01 for 48 h. Viral NP levels were determined with western blotting (E), and densitometrically quantified and normalized to β-actin (F). The progeny virus was determined by TCID₅₀ assay (G). *P<0.05 vs. Con, **P<0.01 vs. Con and ***P<0.001 vs. Con (One-way ANOVA, Bonferroni’s multiple comparisons test).

miR-193b suppresses IAV infection by targeting β-catenin

To determine how miR-193b regulates IAV replication by altering the Wnt/β-catenin signaling pathway, we used PicTar, Target Scan (version 5.2) and DIANA-microT-4.0 to predict the target genes of miR-193b in the pathway. Since miR-193b inhibits Wnt/β-catenin signaling, we narrowed down the target selection to the positive regulators of the signaling. Three positive regulators of the Wnt/β-catenin signaling pathway, LEF1, CTNNB1 and FZD4, were predicted to contain binding sites for miR-193b. FZD4 encodes frizzled 4 protein, which acts as a receptor for Wnt ligands (H. C. Huang & Klein, 2004). LEF1 and CTNNB1 encode the Wnt pathway transcription factor LEF1 and coactivator β-catenin, respectively. Accumulation of β-catenin in the cytoplasm and its eventual translocation into the nucleus act as a transcriptional coactivator of the transcription factor LEF1 (Komiya & Habas, 2008). The potential binding sites between miR-193b and the 3’-UTR of the targets are shown in Fig. 6A. To determine whether miR-193b binds these targets, we cloned the 3’-UTRs of these genes into a luciferase reporter and transfected them into HEK293T cells along with the miR-193b expression plasmid. miR-193b reduced the luciferase activities by 35-50% of all targets (Fig. 6B), suggesting that miR-193b was able to bind the 3’-UTRs of LEF1, CTNNB1 and FZD4.

Fig. 6. miR-193b target verification.

(A) Potential interactions between miR-193b and the putative binding sites in the 3’-UTR of LEF1, β-catenin and FZD4. (B) The 3’-UTR reporter assay. HEK293T cells were transfected with a miR-193b expression vector and a 3’-UTR luciferase reporter plasmid for 48 h. Firefly luciferase activity was normalized to pRL-TK Renilla luciferase activity and then expressed as a ratio to the vector control (miR-Con). (C, D) Effect of miR-193b on endogenous target proteins in HEK293 cells. HEK293 cells were transfected with a miR-193b overexpression plasmid for 48 h. LEF1, β-catenin, and FZD4 protein expression was detected by western blotting, densitometrically quantified and normalized to β-actin. (E, F) Effect of miR-193b on endogenous β-catenin in A549 cells. A549 cells were infected with a miR-193b lentivirus for 48 h. β-catenin protein expression was detected by western blotting, densitometrically quantified and normalized to β-actin. (G, H) Endogenous β-catenin in miR-193b-knockout A549 cells, β-catenin protein expression in sgRNA1, sgRNA2 or control vector-treated A549 cells was determined by western blotting and quantified by normalization to β-actin. Data are expressed as the mean ± SE of three independent experiments. *P<0.05 vs. miR-Con, **P<0.01 vs. miR-Con, ***P<0.001 and ****P<0.0001 vs. miR-Con (B and D: Two-way ANOVA, Bonferroni’s multiple comparisons test; F: Student’s t-test; H: One-way ANOVA, Bonferroni’s multiple comparisons test).

We further examined whether miR-193b reduced endogenous target proteins. HEK293 cells were transfected with the miR-193b plasmid for 48 h, and western blotting was then performed to evaluate the LEF1, β-catenin and FZD4 protein levels. The LEF1 and β-catenin protein levels were reduced by 71 ± 6% and 30 ± 2%, respectively, in miR-193b-overexpressing HEK293 cells. However, endogenous FZD4 was not affected by miR-193b overexpression (Fig. 6C, D). These results suggest that LEF1 and β-catenin, but not FZD4, are the targets of miR-193b in HEK293 cells. Hereafter, we further examined whether miR-193b also targeted LEF1 and β-catenin in A549 cells. We infected A549 cells with an MOI of 100 of miR-193b lentivirus or the control for 48 h and detected LEF1 and β-catenin by western blotting, β-catenin was reduced by overexpression of miR-193b in both of HEK293 and A549 cells (Fig. 6E, F). We were unable to detect LEF1 in A549 cells due to its low basal expression. Furthermore, the endogenous β-catenin was elevated in the miR-193b knock-out A549 cells, but only sgRNA1 reached a significant level (Fig. 6G, H).

To determine whether LEF1 and β-catenin were involved in the anti-IAV activities of miR-193b, rescue experiments were performed by overexpressing target genes using expression vectors that do not contain the 3’-UTR of the target genes. HEK293 cells were co-transfected with miR-193b alone or along with LEF1 or ΔGSK-β-catenin plasmids for 24 h and then infected with A/PR/8/34 (MOI 0.01) for 48 h. ΔGSK-β-catenin expresses a stabilized form of β-catenin that has mutations at four GSK3β phosphorylation sites (Ser33, Ser37, Thr41 and Ser45) (Barth, Stewart, & Nelson, 1999). Consistent with the previous results (Fig. 2), miR-193b reduced endogenous β-catenin protein levels (lower bands) as well as the NP protein levels and virus titers in culture media (Fig. 7A-C). Ectopic expression of β-catenin was also observed in the ΔGSK-β-catenin-transfected cells (higher bands). The co-expression of ΔGSK-β-catenin and miR-193b abolished the miR-193b-mediated reduction of NP protein level and virus titer. However, co-expression of LEF1 with miR-193b was not able to restore the miR-193b-mediated decreases in NP protein level and progeny virus production (Fig. 7D-F). This data suggested that β-catenin was involved in miR-193b-mediated inhibition of IAV replication.

Fig. 7. β-catenin is involved in miR-193b-mediated inhibition of IAV infection.

HEK293 cells were seeded in 12-well plates and transfected with a miR-193b plasmid (0.5 μg) alone or along with pCMV-ΔGSK-β-catenin (β-catenin) (0.5 μg) (A-C) or together with pCS2-LEF-l (0.25 μg) (D-F). The cells were then infected with A/PR/8/34 (MOI 0.01) for 48 h. NP protein expression was detected by western blotting (A and D), quantified, and normalized to β-actin (B and E). Progeny virus was determined by TCID₅₀ assay (C and F). Data are expressed as the mean ± SE of three independent experiments. *P<0.05, **P<0.01 (One-way ANOVA, Tukey’s multiple comparison test). NS: not significant.

miR-193b suppresses cyclin D1 and induces G0/G1 arrest

As a downstream target of Wnt/β-catenin signaling, cyclin D1 plays an important role in the G1-S transition of the cell cycle. Therefore, we further determined whether miR-193b reduced cyclin D1 expression. To this purpose, we transfected HEK293 cells with the miR-193b plasmid or infected A549 cells with miR-193b lentivirus. Real-time PCR and western blotting analyses showed that miR-193b reduced the mRNA and protein levels of cyclin D1 in HEK293 and A549 cells (Fig. 8A-C). To determine whether the restoration of cyclin D1 protein level can overcome the inhibition of IAV infection caused by miR-193b, we transfected HEK293 cells with a cyclin D1 expression vector alone or together with a miR-193b expression vector. The results showed that ectopic expression of cyclin D1 abolished the miR-193b-mediated reduction of IAV infection (Fig. 8D, E, F).

Fig. 8. miR-193b suppresses cyclin D1 and induces G0/G1 cell cycle arrest.

(A-C) HEK293 cells were transfected with a miR-193b expression plasmid for 48 h or A549 cells were infected with a miR-193b lentivirus (MOI 100) for 48 h. The mRNA (A) and protein (B) levels of cyclin D1 were detected by real-time PCR and western blotting, respectively. Protein expression was densitometrically quantified and normalized to β-actin (C). (D-F) HEK293 cells were seeded in 12-well plates and transfected with a miR-193b plasmid (0.5 μg) alone or along together with p-CMV-CCNDl (0.5 μg, Addgene # 19927) expression plasmid. The cells were then infected with A/PR/8/34 (MOI 0.01) for 48 h. NP protein expression was detected by western blotting, quantified, and normalized to β-actin. Progeny virus was determined by TCID₅₀ assay. (G) miR-193b lentivirus-infected A549 cells were serum-starved for 24 h and then cultured in medium containing 10% fetal bovine serum for 18 and 30 h. The cells were stained with propidium iodide. The DNA content was measured by flow cytometry. The cell cycle phase distribution was examined. Data are expressed as the mean ± SE of three independent experiments. A, C and G: *P<0.05 vs. miR-Con, **P<0.01 vs. miR-Con (Two-way ANOVA, Bonferroni’s multiple comparisons test); E and F: *P<0.05 and **P<0.01 (One-way ANOVA, Tukey’s multiple comparison test).

To test whether miR-193b was able to affect the cell cycle progression, miR-193b-infected A549 cells were synchronized by cultivation in serum-free medium for 48 h and then allowed to re-enter the cell cycle by culturing them in medium containing 10% FBS for 18 and 30 h. The cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Approximately 72% of miR-193b-infected cells were in the G0/G1 phase compared with 60-62% of miR-Con-treated cells (Fig. 8G) at both time points. On the contrary, miR-193b overexpression decreased the progression of A549 cells to S phase. This data indicated that miR-193b suppressed cyclin D1 expression and inhibited the cell cycle transition from G0/G1 to S.

miR-193b hinders the nuclear entry of vRNP

IAV infection involves a complex series of nuclear import and export events. Thus, we examined whether miR-193b was able to inhibit vRNP entry into the nucleus. We infected miR-193b-overexpressing A549 cells with A/PR/8/34 for various times and immunostained the cells with NP antibodies. At 2 hpi, 2% of control cells showed a NP signal in the nucleus compared with almost no signal in the miR-193b-overexpressing cells. At 3 hpi, 57 ± 9% of control cells displayed NP nuclear localization compared with only 13 ± 4% of the miR-193b-overexpressing cells. At 5 hpi, the proportions of cells with NP nuclear localization increased to 93 ± 2% in control cells and 55 ± 9% in miR-193b-overexpressing cells (Fig. 9A, B). At 8 h after infection, most of control and miR-193b-overexpressing cells displayed a NP signal centered in the nuclei. This data suggested that miR-193b delayed vRNP entry into the nucleus.

Fig. 9. miR-193b suppresses IAV vRNP nuclear import.

(A) miR-193b or miR-Con lentivirus-infected A549 cells were infected with A/PR/8/34 at an MOI of 10 for 2, 3, 5 and 8 h. Viral NP (red) was immunostained, and the nuclei (blue) were counterstained. (B) Cells with intense NP signal localized (centered) in the nucleus were considered to exhibit nuclear localization and counted. The percent of cells with NP nuclear localization at different times was quantified. Data are expressed as the mean ± SE of three independent experiments. ***P<0.001 vs. miR-Con (Two-way ANOVA, Bonferroni’s multiple comparisons test).

G0/G1 cell cycle arrest inhibits the nuclear entry of vRNP

Since miR-193b induces G0/G1 arrest, we investigated the effect of cell cycle arrest on vRNP nuclear import. G0/G1-arrested A549 cells were obtained by serum starvation in medium containing 0.2% FBS for 72 h. Control cells were initially serum-starved for 48 h and then cultured in complete medium for 24 h, which allowed the cells to re-enter the normal cell cycle. The cells were then infected at an MOI of 10 of A/PR/8/34 virus for 3 h, and immunofluorescent staining was performed to detect viral NP. Ki-67 staining was performed to examine cell cycle arrest. As shown in Fig. 10A and B, the fluorescence signal of Ki-67, a marker for cell proliferation, was significantly reduced in serum-starved cells compared with the control. By contrast, NP nuclear translocation was 2.3-fold higher in control cells than in the cells that were in G0/G1 cell cycle arrest. This data indicated that G0/G1 cell cycle arrest indeed suppressed vRNP entry into the nucleus.

Fig. 10. Effect of G0/G1 cell cycle arrest on IAV NP nuclear import.

G0/G1-arrested A549 cells were achieved by culturing the cells in medium containing 0.2% of fetal bovine serum for 72 h. Control cells were first serum-starved in medium containing 0.2% fetal bovine serum for 48 h and then cultured in complete medium (containing 10% fetal bovine serum) for 24 h to allow them to enter the normal cell cycle. (A) Ki-67 was immunostained (green) and cell nuclei were counterstained (blue). (B) Quantification of Ki-67 positive cells. (C) Control or arrested cells were infected with A/PR/8/34 (MOI of 10) for 3 h. Immunofluorescence staining was performed to detect viral NP (red). The cells were counterstained with Hoechst 33342 (blue). (D) The percent of cells with NP nuclear localization was quantified. Data are expressed as the mean ± SE of three independent experiments. *P<0.05 vs. Control, **P<0.01 vs. Control (Student’s t-test).

IAV infection causes downregulation of miR-193b in vitro

To assess the effect of IAV infection on miR-193b expression, A549 or HEK293 cells were infected with A/PR/8/34 at MOI 0.01 for 0, 24, 48 and 72 h. As determined by real-time PCR, the expression level of miR-193b shows a trend in reduction with time of infection; and reached a significant decrease at 72 h post infection in both A549 and HEK293 cells (Fig. 11), suggesting that IAV infection may counteract the miR-193b expression.

Fig. 11. Effect of IAV infection on miR-193b expression.

A549 (A) or HEK293 (B) cells were infected with A/PR/8/34 (MOI 0.01) for 0, 24, 48 and 72 h. miR-193b expression in the cells was determined by real-time PCR, and was normalized to U48. *P<0.05 vs. 0 h (One-way ANOVA, Bonferroni’s multiple comparisons test).

miR-193b suppresses viral infection in mice challenged with a sublethal dose of A/PR/8/34

To determine whether miR-193b inhibited IAV replication in vivo, we overexpressed miR-193b in the lungs of mice using an adenoviral vector, challenged mice with a sublethal dose of A/PR/8/34 (250 PFU/mouse), and examined the body weight as well as viral mRNA and progeny virus production in lung tissues at day 3 and 7 post infection. No mortality was observed in either control or miR-193b-treated mice. Body weight loss was slightly improved in miR-193b-treated mice (Fig. 12A). As determined by real-time PCR, the miR-193b levels in lung tissues were approximately 22- and 6-fold higher than those in the control at day 3 and 7 post infection, respectively (Fig. 12B). The decrease in miR-193b expression from day 3 to day 7 was likely due to transient expression of miR-193b by the adenovirus vector. Cyclin D1 mRNA was clearly suppressed in the lung of the mice overexpressing miR-193b (Fig. 12C). Viral NP mRNA was significantly reduced by overexpression of miR-193b in the lungs of mice at 7 post infection (Fig. 12D). Progeny virus production in the lungs was also decreased at 7 dpi although it did not reach a significant level (Fig. 12E). Taken together, these results confirm the anti-flu activities of miR-193b in vivo.

Fig. 12. miR-193b suppresses viral infection in mice challenged with a sublethal dose of A/PR/8/34.

Two days after receiving 10⁹ IFU of adenoviral miR-193b or miR-Con, C57BL/6J mice were infected with A/PR/8/34 (250 PFU/mouse). Mice were euthanized at day 3 or 7 post infection. (A) Percent of body weight normalized to the initial body weight (n= 7). (B) miR-193b expression in the lungs of mice at day 3 or 7 post infection was determined by real-time PCR, normalized to β-actin and expressed as a ratio to miR-Con on the corresponding days (n=7). (C) Cyclin D1 mRNA expression was determined by real-time PCR and normalized to β-actin (n=7). (D) Viral mRNA levels were detected by real-time PCR and normalized to β-actin (n=7). (E) Virus progeny production in lung homogenate was determined by TCID₅₀ assay (n=7). The data are presented as the mean ± SD. *P < 0.05 vs. miR-Con and **P < 0.01 vs. miR-Con on the corresponding days (Non-parametric Friedman’s Two-way ANOVA, Bonferroni’s multiple comparisons test).

Discussions

As influenza pathogenesis is determined in part by the host response, understanding the key host modulators of viral infection and virus-induced disease is a promising approach to host-oriented drug development for preventing the disease. The Wnt/β-catenin signaling pathway has been shown to play an important role in virus replication (More et al., 2018; Shapira et al., 2009) and virus-induced immune responses (Hillesheim, Nordhoff, Boergeling, Ludwig, & Wixler, 2014). Identification of miRNAs that interfere with IAV replication by modulating Wnt/β-catenin will provide new insights into the miRNA-Wnt/β-catenin signaling-IAV interaction network. In the present study, using miRNA library screening, we identified 15 and 5 miRNAs that up- and downregulated Wnt/β-catenin signaling, respectively (Fig. 1, Table S1). Furthermore, four miRNAs (miR-193b, miR-548f-1, miR-1-1 and miR-509-1) that downregulated the Wnt/β-catenin signaling pathway were shown to suppress IAV replication and virus production. miR-193b, which reduced Wnt/β-catenin pathway signaling most significantly, showed the highest inhibition of viral mRNA and proteins in vitro (Fig. 2). Furthermore, mice overexpressing miR-193b in the lung by adenoviral delivery benefited from less bodyweight loss and viral load after challenge with a sublethal dose of A/PR/8/34 virus (Fig. 12).

Emerging evidence supports the importance of miRNA-mediated gene regulation of the Wnt/β-catenin signaling pathway. A number of miRNAs regulate Wnt signaling by modulating Wnt ligands, β-catenin, transcription factors and the destruction complex. For example, miR-22, -200b, -185, -26a, -329 and -410 target Wnt ligands to inhibit the Wnt/β-catenin signaling pathway (G. Li et al., 2014; Shiah et al., 2014; Tang et al., 2013; Z. Wang, Humphries, Xiao, Jiang, & Yang, 2014; Zhao et al., 2014). The miR-200 cluster targets β-catenin to inhibit cancer progression (J. Liu et al., 2013; Tian et al., 2014). miR-155 negatively regulates APC in multiple carcinomas, including HCC and papillary thyroid carcinoma (X. Zhang et al., 2013), to promote cell proliferation and tumorigenesis. In the present study, we found that miR-193a/b, miR-548f-1, miR-1-1 and miR-509-1 significantly repressed Wnt/β-catenin signaling. Excluding miR-1, which has been previously reported (Anton et al., 2011), miR-193b, miR-548f-1 and miR-509-1 are newly identified negative regulators of Wnt/β-catenin signaling. miR-1-1 and miR-133a-2 are members of a cluster family. They are encoded at two paralogous loci and are reported to downregulate and display tumor-suppressive functions in various human cancers (Hudson et al., 2012; Kojima et al., 2012; Nasser et al., 2008). miR-509-1, previously named miR-509, inhibits cell proliferation and migration in multiple carcinomas (B. Liu et al., 2015; Ma et al., 2016; W. B. Zhang, Pan, Yang, & Zheng, 2013).

All of the 4 miRNAs that inhibit Wnt/β-catenin signaling reduced viral protein levels. It is noted that to the extent that a miRNA inhibits Wnt/β-catenin signaling does not exactly correlate with its anti-influenza virus activities (Fig. 1F and Fig. 2D). It is likely that, in addition to Wnt/β-catenin signaling, these miRNAs also target other pathways. Among the downregulating miRNAs, miR-193b was the most effective suppressor of the Wnt/β-catenin signaling pathway. miR-193b is considered to be a biomarker of various cancers because it is downregulated in lung cancer (H. Hu, Li, Liu, & Ni, 2012), hepatocellular carcinoma (Xu et al., 2010), prostate cancer (Rauhala et al., 2010) and melanoma (Chen et al., 2010). By contrast, miR-193b has been demonstrated to suppress carcinoma cell proliferation, migration, invasion and metastasis (Leivonen et al., 2009; Wu, Lin, Zhuang, & Liang, 2009), suggesting that miR-193b may function as a tumor suppressor. Excluding hepatitis B virus-associated hepatocellular carcinoma (Mao et al., 2014), the role of miR-193b in modulating viral infection has rarely been studied to date. In this study, we found that miR-193b strongly suppressed IAV infection. We further demonstrated that miR-193b inhibited IAV infection by targeting and repressing β-catenin (Fig. 6 and 7). To the best of our knowledge, this is the first report to show that miR-193b possesses antiviral activities.

During virus infection, various cellular signaling cascades are activated, which may support or inhibit viral replication. One important signaling pathway that modulates viral replication is the canonical Wnt/β-catenin pathway. Two recent genome-wide siRNA screens identified multiple components of the Wnt pathway that interfere with Rift Valley fever virus and flavivirus infection (Harmon et al., 2016; Smith, Jeng, McWeeney, & Hirsch, 2017). Previous studies have also shown that the deletion of Wnt pathway components significantly impacts IAV replication and interferon production (Shapira et al., 2009; Watanabe et al., 2014). β-catenin is a part of adherens junctions at the cell membrane and acts as a transcription factor that interacts with LEF/TCF to regulate the Wnt/β-catenin pathway. Increasing evidence has suggested the importance of β-catenin in viral replication, but some discrepancies exist. Hillesheim et al showed that β-catenin accumulates in the nucleus after IAV infection and that β-catenin supports IRF3-dependent transcription of genes responsible for the cellular innate immune response, such as IFN-β and ISGs (Hillesheim et al., 2014). By contrast, Baril and co-workers reported that infection by SeV, another negative-strand RNA virus, induces secretion of Wnt ligands and stabilization of β-catenin and decreases expression of IFNB1 via a feedback mechanism (Baril et al., 2013). Our study showed that the reduction of β-catenin by miR-193b indeed hampered IAV replication and that compensating β-catenin by β-catenin ectopic expression restored IAV infection (Fig. 7). Similarly, Watanabe et al. showed that downregulation of β-catenin significantly decreased the relative viral RNA polymerase activity by more than 50% (Watanabe et al., 2014). Although the reasons for the discrepancy in regard to the roles of β-catenin in the RNA virus-induced interferon response and virus infection are not known, different posttranslational modifications of the β-catenin protein due to the different Wnt stimuli as well as the different viruses and cell types used might be plausible explanations.

LEF1 is also a target of miR-193b. However, LEF1 co-overexpression with miR-193b plasmid did not rescue the miR-193b-mediated reduction of IAV infection. LEF1 forms a complex with β-catenin to activate the Wnt/β-catenin responsive genes. Two reports have shown that while overexpressing LEF1 alone does not activate the Wnt/β-catenin signaling pathway, co-expression of LEF1 and β-catenin results in 50 to 100-fold increases in the TOPFlash activity (Ahrens, Romereim, & Dudley, 2011; Ishitani, Matsumoto, Chitnis, & Itoh, 2005). It has also been reported that Drosophila TCF activates gene transcription in the presence of Armadillo, the Drosophila homolog of β-catenin. However, Drosophila TCF actually functions as a repressor in the absence of Armadillo (Cavallo et al., 1998; de Lau & Clevers, 2001). This may explain why LEF1 overexpression alone even resulted in a decrease of IAV NP protein expression as well as virus production (Fig. 7D-F). Furthermore, β-catenin has a transactivation domain, but not LEF-1 (Vleminckx, Kemler, & Hecht, 1999). These studies suggest that LEF1 requires β-catenin for its transcription activation activity. Since miR-193b reduces the protein levels of both β-catenin and LEF1, overexpression of LEF1 alone cannot rescue the miR-193b effects on IAV infection.

Because miR-193b inhibits both Wnt/β-catenin signaling and influenza virus infection, we focused our target identification of miR-193b on positive regulators of the Wnt/β-catenin pathway. However, we cannot rule out the possibility that miR-193b may also act on other targets and pathways. There are several reported targets of miR-193b in different cells. For example, miR-193b targets ETS1 transcription factor to cause cell cycle arrest and to inhibit the invasion and migration of hepatoma cells (Xu et al., 2010). Upregulation of c-Kit oncogene frequently occurs in subsets of acute myeloid leukemia (AML). Overexpression of miR-193b in the Kasumi-1 AML cell line decreases c-Kit expression and inhibits the downstream PDKl/Akt signaling pathway (Gao et al., 2011). miR-193b also targets the urokinase-type plasminogen activator (uPA), an enzyme involved in the degradation of extracellular matrix proteins, resulting in the repression of tumor progression and invasion in human breast cancer (X. F. Li, Yan, & Shao, 2009). Last but not the least, miR-193b induces G1-phase arrest in pancreatic ductal adenocarcinoma by targeting KRAS, through which the AKT and ERK pathways were modulated (Jin et al., 2015).

A good number of studies suggest that crosstalk occurs between the cell cycle and Wnt signaling. It has been well demonstrated that Wnt/β-catenin signaling stimulates the G1/S transition and promotes cell cycle progression and therefore proliferation through transcriptional upregulation of target genes that encode, for example, c-Myc and cyclin D (Niehrs & Acebron, 2012). By contrast, the β-catenin levels increase during the G1/S transition and peak during G2/M in normal and transformed epithelial cells (Olmeda, Castel, Vilaro, & Cano, 2003). It was recently found that the mitotic CDK14/cyclin Y complex promotes Wnt/β-catenin signaling through phosphorylation of the LRP6 co-receptor (Davidson & Niehrs, 2010). Cyclin D1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle and is necessary for cells to enter S phase (Baldin, Lukas, Marcote, Pagano, & Draetta, 1993). Downregulation of cyclin D1 induces G0/G1 arrest (Masamha & Benbrook, 2009; Radu, Neubauer, Akagi, Hanafusa, & Georgescu, 2003). We observed that cyclin D1 is markedly downregulated in miR-193b-overexpressing cells. This is likely due to the combined effect of a reduced Wnt/β-catenin signaling activity and a direct repression by miR-193b because cyclin D1 is a well-known down-stream target of Wnt/β-catenin signaling (Shtutman et al., 1999) and cyclin D1 has been previously shown to be a direct target of miR-193 in cancer cells (Chen et al., 2010).

Based on the observations that miR-193b reduced cyclin D1 protein levels and induced cell cycle G0/G1 arrest, and that miR-193b and cell cycle G0/G1 arrest suppressed viral vRNP nuclear import, we speculate that miR-193b may inhibit vRNP nuclear import by minimizing the cell cycle conditions needed for efficient viral nucleoprotein expression and/or transport. The correlation of the cell cycle and viral replication has been reported. For instance, some DNA viruses promote quiescent cells entry into S phase to support viral genome synthesis. By contrast, some large DNA viruses and RNA viruses can arrest cells in a particular phase of the cell cycle that is favorable for replication of a specific virus (Bagga & Bouchard, 2014; Emmett, Dove, Mahoney, Wurm, & Hiscox, 2005). Another mechanism may be related to cellular traffic. For example, Cawood et al demonstrated that the nucleocapsid protein of Coronavirus is more mobile in proliferative cells and localizes to the nucleolus in a cell cycle-dependent manner (Cawood, Harrison, Dove, Reed, & Hiscox, 2007). The trafficking of viral proteins may depend on the dynamics of the nucleolar proteome in response to the metabolic profile of the cell. It is noted that two studies suggest that cell cycle G0/G1 arrest facilitates IAV infection (He et al., 2010; Jiang et al., 2013).

In summary, herein, we identified several miRNAs as novel negative regulators of Wnt/β-catenin signaling. miR-193b possesses potent anti-influenza virus activities. The inhibitory effect of miR-193b on IAV infection occurs through inhibition of β-catenin, likely by inducing cell cycle arrest. This discovery may facilitate the identification of potential targets for anti-influenza therapeutics and drug discovery.

Experimental Procedures

Cells and viruses

Human lung epithelial A549 cells (American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained in F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S). Madin-Darby Canine Kidney (MDCK, ATCC) epithelial, human embryonic kidney (HEK) 293 and HEK293T (ATCC) cells were maintained in DMEM containing 10% FBS and 1% P/S.

Influenza A/Puerto Rico/8/34 (A/PR/8/34) H1N1 virus, a well-characterized mouse adapted laboratory strain, and influenza 2009 pandemic clinic isolate A/Oklahoma/3052/09 (OK/09) H1N1 virus were propagated in the allantoic cavity of 10-day-old embryonated chicken eggs (Charles River Laboratories, MA, USA). Influenza A/WSN/1933 (A/WSN/33) H1N1 virus and A/Oklahoma/309/2006 (A/Oklahoma/309/06) H3N2 virus were propagated in MDCK cells.

Cell viability assay

Cell viability was determined by using a CellTiter Blue Cell Viability assay kit (Promega, Madison, Wl, USA) according to the manufacturer’s instructions. Cells were cultured in 96-well plates and were incubated with the CellTiter Blue Reagent at 37°C for 2 h. Fluorescence was measured using an excitation at 560 nm and an emission at 590 nm on a plate reader.

miRNA expression library construction

The miRNA overexpression vectors contained the CMV promoter, followed by an enhanced green florescent protein (EGFP) tag, a mature miRNA with flanking sequences (~ 200 bp at each end), and the SV40 poly(A) terminal sequence. Mature miRNAs plus flanking sequences were amplified from human genomic DNAs and cloned via Xho I and EcoR I sites into a modified pLVX-Puro lentiviral vector (Clontech, Mountain View, CA, USA) or a modified pENTR vector between EGFP and SV40 poly(A) terminal sequence, as previously described (Bhaskaran et al., 2009; C. Huang et al., 2017). Inserted vector with a similar size of genomic DNA that did not contain any miRNAs or stem loop structures was used as a control (miR-con).

High-throughput miRNA screening

A TOPflash reporter vector containing three copies of T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) binding sites upstream of the thiamine kinase minimal promoter and firefly luciferase gene (Upstate Biotechnology, Inc., Lake Placid, NY) was used to monitor the Wnt/β-catenin activity. HEK293T cells were seeded in antibiotic-free culture medium in 96-well plates (2 × 10⁴ cells/well). At 20 h after seeding, the cells were transfected with a TOPflash vector (20 ng), a pLVX-Puro lentiviral miRNA or control plasmid (miR-Con) (100 ng), and a normalization pRL-TK vector (0.2 ng), using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). At 20 h post-transfection, the cells were stimulated with Wnt3a conditioned medium (50%) (Guo et al., 2014) for 24 h and lysed in 70 μl passive lysis buffer (Promega, Madison, WI, USA) for 20 min. After a brief centrifugation (12,000 g for 5 min at 4°C), the supernatant was collected. Forty microliters of firefly and 40 μl of Renilla luciferase reagents were dispensed into each well of a 96-well plate, in which 10 μl of the collected supernatant had been previously added. Readings were collected using a plate luminometer (BMG LABTECH, Cary, NC, USA). Primary screening was performed in triplicate in a 96-well plate. Firefly luciferase activities in lysates were normalized to those of Renilla luciferase. The results are expressed as ratios of the relative luciferase activities over miR-Con. To verify the results, the positive hits from the initial screening were screened for the second time using a TOPflash vector and a FOPflash containing the mutated LEF-1 binding sites under the same conditions as in the primary screening.

MicroRNA target prediction

The bioinformatics prediction tools PicTar (http://pictar.mdc-berlin.de/), Target Scan (version 5.2) (http://www.targetscan.org/) and DIANA-microT-4.0 (http://diana.imis.athena-innovation.gr/) were used to identify potential targets of miR-193b.

Construction of 3’-UTR luciferase reporter vectors and the IAV reporter vector

The 3’-UTRs of predicted target genes of the miRNAs were PCR-amplified and inserted into the pmirGLO dual-luciferase miRNA target expression vector (Promega, Madison, WI) at the NheI and SalI sites.

To detect IAV infection with a luciferase reporter assay, we constructed an IAV reporter vector encoding firefly luciferase under control of the 3’-UTRs of the influenza A/WSN/33 NP segment as previously described (Lutz, Dyall, Olivo, & Pekosz, 2005). Briefly, the 3’- and 5’-UTRs of the influenza A/WSN/33 NPs were amplified by PCR and inserted into the RNA polymerase I promoter/terminator cassette of the pHH21 vector (a kind gift from Dr. Yoshi Kawaoka, U. Wisconsin) at BsmB I sites (pHH21-NP-3’-UTR-LUC-NP-5’-UTR).

All inserts were confirmed by DNA sequencing.

Production of miRNA lentivirus or adenovirus

A lentiviral vector was co-transfected with psPAX2 and pMD2G (Cellecta, Mountain View, CA, USA) into HEK293T cells using polyethylenimine (PEI, Sigma, St. Louis, MO, USA) (6 μg PEI/μg plasmid) to generate lentivirus expressing miR-193b or the control. The pENTR-miRNA vector or miR-con was switched into an adenovirus vector using the Gateway technique pAd/CMV/V5-DEST^™ (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. Adenoviral vectors were then linearized with PacI before they were transfected to HEK293A cells. The virus was harvested and further amplified by re-infecting HEK293A cells. The adenovirus was eventually purified using the Adeno-X^™ Maxi Purification Kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. Infectious units (IU) of both lenti- and adeno-miRNA viruses were determined by titration in HEK293T cells.

IAV infection

HEK293 cells were seeded in a 12-well plate (5 × 10⁵ cells/well) and then transfected with 1.25 μg of pENTR-miRNA or miR-Con vector using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h. A549 cells were seeded in a 12-well plate (2 × 10⁵ cells/well) and then infected with miR-193b or miR-Con lentivirus at a multiplicity of infection (MOI) of 100 for 48 h. Thereafter, the cells were infected with IAV at the indicated MOIs in serum-free medium containing 0.5 μg/ml of trypsin treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK, Worthington Biochemical Corporation, Lakewood, NJ, USA). At 1 h post-viral absorption, the inoculum was removed, and serum-free medium containing 0.3% bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) and 0.5 μg/ml of TPCK-treated trypsin was added and incubated for the indicated times. The cells were collected for mRNA and protein analyses, and the culture media were collected for titer determination.

Median tissue culture infective dose (TCID₅₀) assay

Virus titrations were performed according to a protocol adapted from De Vleeschauwer A. et al (De Vleeschauwer, Van Poucke, Karasin, Olsen, & Van Reeth, 2011). Briefly, MDCK cells were seeded in a 96-well cell culture plate at a density of 20,000 cells per well. One day after seeding, the cells were inoculated with 50 μl of 10-fold serially diluted samples. After 1 h, the inoculum was removed, followed by the addition of serum-free DMEM containing 1 μg/ml TPCK-trypsin. The cells were observed daily for 5 days for the appearance of cytopathic effects (CPE). Virus titers were calculated according to the method of Reed and Muench (Reed LJ, 1938).

Reverse transcription and real-time PCR

To measure mRNA expression levels, total RNA was extracted using TRI Reagent^® (Molecular Research Center, Cincinnati, OH, USA). One microgram of RNA was treated with RNase-Free DNase I (Thermo Scientific, Waltham, MA, USA) at 37°C for 30 min and then reverse-transcribed using M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) with oligo(dT)18 and random hexamer primers (Promega, Madison, Wl, USA). Real-time PCR was carried out in a 20-μl reaction volume containing specific primers (Table 1) and SYBR Green PCR Master Mix (Eurogentec, Fremont, CA, USA). PCR was performed on an ABI 7500 real-time PCR System (Applied Biosystems, Foster City, CA, USA). The cycling conditions for real-time PCR were as follows: 95°C for 1 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression level of a gene was normalized to the internal control, β-actin, and the expression level was calculated using the 2^−ΔCt method.

Table 1.

Real-time PCR primers

Primer name	Forward primers 5’-3’	Reverse primers 5’-3’
hsa-miR-193b	AACTGGCCCTCAAAGTCCCG	GCGAGCACAGAATTAATACGAC
hsa-miR-548f-1	TGCAAAAGTAATCACAGTT	GCGAGCACAGAATTAATACGAC
hsa-miR-1-1	TGGAATGTAAAGAAGTATGTAT	GCGAGCACAGAATTAATACGAC
Has-miR-509-1	TGATTGGTACGTCTGTGGGTAG	GCGAGCACAGAATTAATACGAC
PolyT adapter	GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN (N=A+C+G+T, V=G+A+C)
NP	TGTGTATGGACCTGCCGTAGC	CCATCCACACCAGTTGACTCTTG
NS1	TGGAAAGCAAATAGTGGAGCG	GTAGCGCGATGCAGGTACAGAG
PB1	CCGATGCCATAGAGGTGACA	GGAGACCAGCAGTCCAGCTTT
LEF1	GCTTTATCCAGGCTGGTCTGCAA	GACCTGTACCTGATGCAGATTCCT
CTNNB1	CATCTACACAGTTTGATGCTGCT	GCAGTTTTGTCAGTTCAGGGA
FZD4	TTTCACACCGCTCATCCAGTAC	GGGTTCACAGCTCTCTTGACT
CCND1	GCAGACCTTCGTTGCCCTCT	GCGTGTGAGGCGGTAAGTAGG
c-myc	TGGTCGCCCTCCTATGTTG	CCGGGTCGCAGATGAAACTC

Real-time PCR for miRNA expression

Total RNA was extracted using TRI Reagent^® and then polyadenylated using the Poly(A) Polymerase Tailing Kit (NEB, MA, USA). Briefly, a 10-μl reaction containing 1 μg total RNA, 1 μl of 10 × reaction buffer, 1 μl of 10 mM ATP and 5 units of E. coli Poly(A) Polymerase was incubated at 37°C for 20 min, followed by enzyme inactivation at 65°C for 20 min. After polyadenylation, reverse transcription was performed using 10 μl of the polyadenylated reaction product, 1 μl of 0.5 μM Poly(T) adapter, 1 μl of 10 mM dNTP, and 1 μl of M-MLV Reverse Transcriptase. The reaction was incubated at 37°C for 50 min and then terminated by heating at 70°C for 15 min. Real-time PCR were performed using SYBR green PCR master mix with the above-described conditions. The expression of a miRNA was analyzed according to the comparative Ct method by normalization to U48 or U6 small nuclear RNA using the 2^−ΔCt method.

Luciferase reporter assay

For the 3’-UTR reporter assay, HEK293T cells were transfected with 5 ng of 3’-UTR reporter vector, 100 ng of miRNA or miR-con vector using Lipofectamine 2000. Two days after transfection, the cells were harvested for the dual luciferase activity assay.

To determine the effect of miR-193b on different strains of IAV replication, a viral luciferase reporter assay was performed. Briefly, HEK293 or A549 cells were transfected with an IAV reporter vector pHH21-NP-3’-UTR-LUC-NP-5’-UTR (20 ng), a miR-193b pENTR or miR-con vector (100 ng), and a normalization pRL-TK vector (2 ng) using Lipofectamine 3000. Twenty-four hours post-transfection, the cells were infected with different strains of IAV, A/PR/8/34 (MOI 0.01), OK/09 (MOI 0.01), A/WSN/33 (MOI 0.005) or A/Oklahoma/309/2006 (MOI 0.01) for 48 h. The cells were lysed, and the dual luciferase assay was performed. The reporter activity was expressed as a ratio of firefly to Renilla activities, which was further normalized to miR-con.

Western blot analysis

The cells were lysed with an appropriate volume of protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing phosphatase and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was then determined using the Bio-Rad Protein Assay Kit (Bio-Rad, CA, USA). Ten micrograms of each protein sample were electrophoresed by SDS-PAGE and transferred onto a nitrocellulose membrane. Western blot analysis was performed using the following primary antibodies and dilutions: mouse anti-NP (ATCC, HB-65, 1:50), mouse anti-NS1 (Santa Cruz biotechnology, Santa Cruz, CA, USA, 1:1000), mouse anti-β-actin (Thermo Fisher Scientific, 1:3000), rabbit anti-β-catenin (Cell Signaling, Beverly, MA, USA, 1:1000), rabbit anti-LEF1 (Cell signaling, 1:1000), and rabbit anti-FZD4 (Cell signaling, 1:1000). The membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, PA, USA, 1:2000) or goat anti-rabbit IgG (Jackson ImmunoResearch, 1:2000) at room temperature for 1 h. Protein bands were visualized using the enhanced chemiluminescence kit (Thermo Fisher Scientific, Rockford, IL, USA).

Immunofluorescent staining (IF)

For viral NP IF staining, lenti-miR-193b or vector control-infected A549 cells were seeded in a 96-well plate and then infected with A/PR/8/34 virus at an MOI of 10. The cells were then fixed for 20 min in 4% paraformaldehyde and permeabilized for 20 min with 0.2% Triton X-100. Next, the cells were incubated with mouse anti-NP primary IgG antibodies (ATCC, HB-65, 1:20) in PBS containing 10% normal goat serum for 1 h at 37°C, followed by Alexa Flour 546-conjugated goat anti-mouse IgG secondary antibodies (Life technologies, Carlsbad, CA, 1:300) in PBS containing 10% normal goat serum for 1 h at 37°C. Cell nuclei were stained with 2 μg/ml of Hoechst 33342 dye (Molecular probes, Waltham, MA). The fluorescence signal was examined with a Nikon fluorescence microscope. The total number of NP nuclear-centered cells and the total number of cells were counted. Data were then presented as percentages of NP nuclear-localized cells over total cells in miR-193b-infected cells vs. miR-Con infected cells.

sgRNA design and generation of the miR-193b knockout cell lines

Two single guide RNAs (sgRNAs) were designed by using online software (http://chopchop.cbu.uib.no/). The PAM sequence near the miR-193b seed sequence and the 20-bp sequence upstream were chosen. We constructed the lentiviral CRISPR/Cas9 mediated miR-193b gene editing vectors by subcloning each annealed sgRNA oligonucleotide pair into the sgRNA scaffold of lentiCRISPR v2 vector (Addgene, #52961) via the Bsmll sites. A control vector was constructed by inserting a EGFP sgRNA sequence into the same vector. Lentivirus was prepared by transfecting sgRNA-lentiCRISPR v2 vector and package plasmids pSPAX2 and pMD2G into HEK293T cells using PEI reagent. Stable cell lines were generated by infecting A549 cells with the CRISPR/Cas9 lentivirus containing sgRNA and selected with 1 μg/ml puromycin.

To detect Indels, genomic DNA was isolated from the CRISPR/Cas9-edited cells. and the targeted locus that encompasses the mature miR-193b region was PCR-amplified using the following primers: Forward 5’AGCTTTGGTCATCAAAATAGGA3’, Reverse 5’ATTGGAGTTTATCGGCAACTGT3’. The PCR products were denatured, annealed and digested with T7 endonuclease I (NEB, Ipswich, MA, USA), which recognizes and cleaves non-perfectly matched DNA, at 37°C for 60 min. Digested products were separated on a 1% agarose gel for imaging and cloned into the pGEM-T vector (Promega, Madison, WI, USA) for DNA sequencing.

Cell cycle analysis

A549 cells were infected with a miR-193b lentivirus or control virus (MOI 100) for 48 h. The cells were trypsinized and then seeded in a 12-well plate (10⁵ cells/well). At 12 h after seeding, the medium was replaced with serum free F-12K and cultured for 48 h. The medium was then replaced with F-12K containing 10% FBS and cultured for 18 h and 30 h. The cells were trypsinized and fixed with ethanol at −20°C for at least 2 h. At the time of analysis, the cells were centrifuged and stained with a freshly made solution containing propidium iodide (PI) provided with the cell cycle phase determination kit (Cayman, Ann Arbor, MI, USA), according to the manufacturer’s instructions. The cell cycle distribution was measured using a flow cytometer (Accuri C6, BD Biosciences) and analyzed with BD Sampler Software.

Animal studies

The animal procedures were approved by the Institutional Animal Care and Use Committee at Oklahoma State University. miR-193b adenovirus or control virus (10⁹ IFU) in 50 μl of PBS were delivered via intratracheal inoculation to the lungs of eight-week-old female C57BL/6J mice 2 days before IAV challenge. IAV Infection was performed by intranasal inoculation of 250 PFU of A/PR/8/34 in 50 μl of PBS. Body weight loss was monitored daily. Mice were euthanized at day 3 or 7 post infection. The left lungs of mice were fixed in 10% neutral-buffered formalin (Thermo Scientific, West Palm Beach, FL, USA). The right lungs were snap-frozen in liquid nitrogen and powered under liquid nitrogen using a mortar and pestle. The powder was divided into three parts: one dissolved in TRI Reagent^® for RNA isolation, one in protein lysis buffer for western blot analysis, and one in DMEM (100 mg/ml) for determination of the viral titer.