Cellular microRNA miR-181b inhibits replication of mink enteritis virus by repression of non-structural protein 1 translation

Abstract

Mink enteritis virus (MEV) is one of the most important viral pathogens in the mink industry. Recent studies have showed that microRNAs (miRNAs), small noncoding RNAs of length ranging from 18-23 nucleotides (nt) participate in host-pathogen interaction networks; however, whether or not miRNAs are involved in MEV infection has not been reported. Our study revealed that miRNA miR-181b inhibited replication of MEV in the feline kidney (F81) cell line by targeting the MEV non-structural protein 1 (NS1) messenger RNA (mRNA) coding region, resulting in NS1 translational repression, while MEV infection reduced miR-181b expression. This is the first description of cellular miRNAs modulating MEV infection in F81 cells, providing further insight into the mechanisms of viral infection, and may be useful in development of naturally-occurring miRNAs antiviral strategies.

Figure 1. Screening of miRNAs that changed more than 2-fold on MEV infection targeting MEV mRNAs.

RNAhybrid and RegRNA tools were used to predict miRNA target sites on MEV mRNAs following the rules of no mismatch and G/U complementary in miRNA seed sequences.

Figure 2. Cellular endogenous miR-181b inhibits MEV production.

(A) TCID₅₀ values were used to assess effects of miR-181b mimics and inhibitors on viral growth curves in F81 cells. NC mimics and inhibitors were used as controls. F81 cells were transfected with mimics or inhibitors for 12 h, and infected with MEV at an MOI of 0.1. Cells were collected at the indicated times and assayed. (B) qPCR was used to assess the effects of miR-181b mimics and inhibitors of MEV genomic DNA at the indicated times from A). The experiments were performed as in A). (C) F81 cells upon infection with MEV from A) were detected through flow cytometric analysis at 36 h post MEV infection, with nonspecific rabbit polyclonal antibody (iso) as an isotype control. (D) F81 cells from A) were observed for development of CPE by bright-field microscopy at 36 h post MEV infection. White scale bar: 50 µm. (E) F81 cells upon infection with MEV from A) were also observed by fluorescence microscopy through indirect immunofluorescence assay at 36 h post MEV infection. White scale bar: 50 µm. Data are representative of 3 independent experiments (mean ± SD). Statistical significance was analyzed by Student’s t test; * P<0.05; ** P<0.01; ns, not significant.

Figure 3. Cellular miR-181b directly targets MEV NS1 mRNA coding region.

(A) Schematic layout of the MEV genome DNA, NS1 mRNA and presumptive target of miR-181b via the 9 sequential complementary nucleotides as indicated. The target and tetranucleotide mutant target segment was cloned into pGL3-control reporter vector. (B) Luciferase activity of lysates of F81 cells after 36 h co-transfection with pGL3-181t, pRL-TK and miR-181b or miR-181c mimics or inhibitors. NC mimics and inhibitors were used as controls. (C) As in B), except that mut pGL3-181t, mut-1 miR-181b mimics and inhibitors were used for transfection. (D) Luciferase activity of lysates of F81 cells infected with MEV at an MOI of 0.1 or transfected with pcDNA-NS1 (0.1 µg/well), and co-transfected with reporter constructs (0.1 µg/well) and miRNA mimics (10 pmol/well) in 24-well plates. Data are from 3 independent experiments (mean ± SD). Statistical significance was analyzed by Student’s t test; *P<0.05; **P<0.01; ***P<0.001; ns, not significant.

Figure 4. Cellular miR-181b inhibits MEV NS1 expression resulting in translational repression through targeting the predicted site of MEV NS1 mRNA.

(A) Western blot assay of lysates of F81 cells after 36 h co-transfection with pcDNA-NS1 and either mimics or inhibitors. (B) qPCR was used to assess effects of miR-181b on the relative expression of NS1 in pcDNA-NS1 with β-actin as an internal control. The experiments were performed as in A). (C) Schematic layout of mut pcDNA-NS1 vector. The nucleotide sequence of the presumptive target, but not the amino acid sequence, of the NS1 gene was altered. Altered nucleotides are underlined. The corresponding mut-2 miR-181b was synthesized. (D) Western blot assay was performed as in A) except that mut pcDNA-NS1 was used instead of pcDNA-NS1. (E) Western blot assay of F81 cells after 36 h co-transfection with mut pcDNA-NS1 and mut-2 miR-181b mimics with NC mimics as control. Data are from 3 independent experiments (mean ± SD). Statistical significance was analyzed by Student’s t test; *P<0.05; ns, not significant.

Figure 5. Cellular miR-181b inhibits MEV replication through targeting the predicted site of MEV NS1 mRNA.

(A) Schematic layout of mut pMEV. The nucleotide sequence of the presumptive target, but not the amino acid sequence, of the NS1 gene was altered. Altered nucleotides are underlined. The corresponding mut-2 miR-181b was synthesized. (B) TCID₅₀ values were used to assess effects of miR-181b mimics and inhibitors or mut-2 miR-181b mimics on viral growth curves of mut MEV in F81 cells. NC mimics and inhibitors were used as controls. F81 cells were transfected with mimics or inhibitors for 12 h and then infected with mut MEV (MOI = 0.1). Cells were collected at the indicated times and titrated. (C) qPCR was used to assess the effects of miR-181b mimics and inhibitors or mut-2 miR-181b mimics of mut MEV genomic DNA at the indicated times from B). (D, E) F81 cells upon infection with mut MEV from B) were detected by flow cytometric analysis at 36 h post MEV infection, with nonspecific rabbit polyclonal antibody (iso) as an isotype control. (F, G) F81 cells from B) were observed for development of CPE by bright-field microscopy at 36 h post MEV infection. White scale bar: 50 µm. (H, I) F81 cells upon infection with mut MEV from B) were also observed by fluorescence microscopy using an indirect immunofluorescence assay. White scale bar: 50 µm. Data are from 3 independent experiments (mean ± SD). Statistical significance was analyzed by Student’s t test; * P<0.05; ** P<0.01; ns, not significant.

Figure 6. miR-181b physically binds to MEV NS1 mRNA in the RISC.

(A) Western blot assay was used to detect Ago2 protein from Ago2 immunoprecipitates of lysates from F81 cells. (B) qPCR analysis of the relative level of miR-181b and MEV NS1 mRNAs from Ago2 or IgG immunoprecipitated lysates of F81 cells transfected with miR-181b mimics (50 pmol/well) in 6-well plates and 12 h later infected with MEV (MOI = 0.1), using U6 small RNA as an internal control. (D) qPCR analysis of the relative level of endogenous miR-181b and MEV NS1 mRNAs from Ago2 or IgG immunoprecipitated lysates without transfection with the mimics. Data are from 3 independent experiments (mean ± SD). Statistical significance was analyzed by Student’s t test; * P<0.05; ** P<0.01.

Figure 7. MEV infection regulates cellular miRNAs including miR-181b.

(A) Expression of selected miRNAs in F81 cells infected with MEV (MOI = 1) and in uninfected controls were tested for validation by qPCR using a specific primer for each miRNA. Fold increase/decrease was calculated based on endogenous control U6 small RNA normalization. (B) qPCR was used to detect the expression levels of miR-181b at 0 h, 6 h, 12 h, 24 h and 36 h after MEV infection, normalized to U6 small RNA. Data are from 3 independent experiments (mean ± SD). Statistical significance was analyzed by Student’s t test; * P<0.05; ** P<0.01; *** P<0.001.