MicroRNA gga-miR-130b Suppresses Infectious Bursal Disease Virus Replication via Targeting of the Viral Genome and Cellular Suppressors of Cytokine Signaling 5

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally through silencing or degrading their targets, thus playing important roles in the immune response. However, the role of miRNAs in the host response against infectious bursal disease virus (IBDV) infection is not clear. In this study, we show that the expression of a series of miRNAs was significantly altered in DF-1 cells after IBDV infection. We found that the miRNA gga-miR-130b inhibited IBDV replication via targeting the specific sequence of IBDV segment A and enhanced the expression of beta interferon (IFN-β) by targeting suppressors of cytokine signaling 5 (SOCS5) in host cells. These findings indicate that gga-miR-130b-3p plays a crucial role in host defense against IBDV infection.IMPORTANCE This work shows that gga-miR-130b suppresses IBDV replication via directly targeting the viral genome and cellular SOCS5, the negative regulator for type I interferon expression, revealing the mechanism underlying gga-miR-130-induced inhibition of IBDV replication. This information will be helpful for the understanding of how host cells combat pathogenic infection by self-encoded small RNA and furthers our knowledge of the role of microRNAs in the cell response to viral infection.

Keywords: IBDV; SOCS; microRNA; type I IFN.

FIG 1

Infection of DF-1 cells with IBDV strain Lx enhances gga-miR-130b expression. (A) KEGG pathway enrichment analysis of miRNAs that were differentially expressed in DF-1 cells upon IBDV infection. The major antiviral pathways in which these miRNAs participated were noted and analyzed. The percentage was calculated as follows: number of miRNAs involved in the cytokine-cytokine receptor, TLR, RLR, or JAK-STAT pathway/total number of miRNAs that participated in these four antiviral pathways. (B) Expression of gga-miR-130b in DF-1 cells with IBDV infection at different time points. DF-1 cells were infected with IBDV at an MOI of 0.1 or mock infected. At different time points (12, 24, 36, and 48 h) after IBDV infection, total RNA was extracted and qRT-PCR was performed to detect gga-miR-130b transcripts. The relative level of gga-miR-130b expression was calculated as follows: expression of miR-130b in IBDV-infected or normal cells/expression of miR-130b in normal cells at 12 h. (C) Expression of gga-miR-130b in DF-1 cells infected with IBDV at different doses. DF-1 cells were infected with IBDV (at an MOI of 0.01, 0.1, 1, or 10) or mock infected. Twenty-four hours after infection, total RNA was extracted and qRT-PCR was performed to detect gga-miR-130b transcripts. The relative level of gga-miR-130b expression was calculated as follows: expression of miR-130b in IBDV-infected cells/expression of miR-130b in normal cells. The expression of U6 snRNA was used as an internal control. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01.

FIG 2

gga-miR-130b enhances poly(I·C)/IBDV-induced expression of type I interferon. (A to D) DF-1 cells were transfected with miRNA controls or miR-130b mimics at 80 nM. Eighteen hours after transfection, cells were infected with IBDV at an MOI of 0.1 or treated with poly(I·C) at a final concentration of 2 μg/ml. Twelve hours after poly(I·C) treatment or IBDV infection, mRNA expression of IFN-α (A), IFN-β (B), IRF3 (C), and NF-κB-p65 (D) was measured by qRT-PCR, using specific primers. The relative levels of gene expression were calculated as follows: mRNA expression of IFN-α, IFN-β, p65, or IRF3 in miR-130b-transfected or normal cells treated with poly(I·C) or IBDV/mRNA expression of miRNA control-transfected cells in control medium. The expression of GAPDH was used as an internal control. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 3

Knockdown of endogenous gga-miR-130b by inhibitors reduces poly(I·C)/IBDV-induced expression of type I interferon. (A to D) DF-1 cells were transfected with miR-130b inhibitors or miRNA inhibitor controls at 80 nM. Eighteen hours after transfection, cells were infected with IBDV at an MOI of 0.1 or treated with poly(I·C) at a final concentration of 2 μg/ml. Twelve hours after poly(I·C) treatment or IBDV infection, cells were harvested for quantification of the expression of IFN-α (A), IFN-β (B), IRF3 (C), and NF-κB-p65 (D). The relative levels of gene expression were calculated as follows: mRNA expression of IFN-α, IFN-β, p65, or IRF3 in miR-130b inhibitor-transfected cells or normal controls treated with poly(I·C) or IBDV/mRNA expression of miRNA inhibitor control-transfected cells in control medium. GAPDH was used as an internal control. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 4

gga-miR-130b inhibits IBDV protein expression. (A and B) Transfection of miR-130b in DF-1 cells reduced expression of VP3 but not VP5. DF-1 cells were transfected with miRNA controls or miR-130b mimics at different doses, followed by infection with IBDV at an MOI of 0.1. Twenty-four hours after infection, cell lysates were prepared and examined by Western blotting using anti-VP3 or anti-VP5 antibodies. Endogenous β-actin expression was used as an internal control. The band densities for VP3 and VP5 in panel A were quantitated by densitometry as shown in panel B. The relative levels of VP3 or VP5 were calculated as follows: band density of VP3 or VP5/band density of β-actin. (C and D) Inhibition of endogenous gga-miR-130b enhanced IBDV VP3 protein expression. DF-1 cells were transfected with miR-130b inhibitors or miRNA control inhibitors before being infected with IBDV at an MOI of 0.1. Twenty-four hours after infection, cell lysates were prepared and examined by Western blotting using anti-VP3 or anti-VP5 antibody. Endogenous β-actin expression was used as an internal control. The band densities of VP3 and VP5 in panel C were quantitated by densitometry as shown in panel D. The relative levels of VP3 and VP5 were calculated as described above. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001.

FIG 5

gga-miR-130b inhibits IBDV replication. (A to F) Examination of IBDV replication by IFA. DF-1 cells were transfected with miRNA controls or miR-130b mimics at 80 nM for 18 h, followed by infection with IBDV Lx at an MOI of 0.1. Twenty-four hours after infection, cells were fixed and examined for the expression of IBDV VP3 protein by immunofluorescent-antibody assays. The pictures in panels A to C were taken under a fluorescence microscope, and those in panels D to F were taken under a light microscope. Magnification, ×100. (G) Analysis of the effect of miR-130b on IBDV replication by TCID₅₀ assay. DF-1 cells were transfected with miRNA controls, miR-130b mimics, or medium only. Eighteen hours after transfection, cells were infected with IBDV Lx at an MOI of 0.1. At different time points (12, 24, 48, and 72 h) after IBDV infection, the viral loads in the cell cultures were determined by TCID₅₀ assays in 96-well plates. The significance of the differences between miR-130b-transfected cells and controls was determined by ANOVA (P < 0.05). The graph shows the average viral loads in DF-1 cells for three individual experiments.

FIG 6

gga-miR-130b directly targets the IBDV genome. (A) Diagram of predicted target sites for miR-130b in IBDV genomic RNA. The seed sequence of miR-130b is underlined and was mutated as indicated by the arrow. (B) miR-130b inhibited target gene expression in a dose-dependent manner. DF-1 cells were cotransfected with luciferase reporter vectors containing wild-type (WT) target sites, pRL-TK, and miR-130b at different concentrations. At 48 h posttransfection, cells were lysed and luciferase reporter gene assays were performed to measure luciferase activities. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miR-130b mimics/luciferase activity of cells cotransfected with the reporter plasmid and miRNA controls. (C) A point mutation in the target gene abolished miR-130b-induced suppression of the target gene. DF-1 cells were cotransfected with miR-130b and the WT or mutant luciferase reporter vector. At 48 h posttransfection, a luciferase reporter gene assay was performed to measure luciferase activity. The relative level of luciferase activity was calculated as follows: luciferase activity of cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 7

The SOCS5 gene is a cellular target of gga-miR-130b. (A) Diagram of predicted target sites for miR-130b in the SOCS5 gene. The seed sequence of miR-130b is underlined and was mutated as indicated by the arrow. (B) Transfection of gga-miR-130b reduced expression of SOCS5. DF-1 cells were cotransfected with miRNAs and luciferase reporter vectors. At 48 h posttransfection, cells were lysed, and a luciferase reporter gene assay was performed to measure SOCS5 expression. The relative level of luciferase activity was calculated as follows: luciferase activity of reporter plasmid-transfected cells or cells cotransfected with the reporter plasmid and miRNA mimics/luciferase activity of cells cotransfected with the WT reporter plasmid and miRNA controls. (C) Mutation of the target site abolished the inhibition of SOCS5 by miR-130b. DF-1 cells were cotransfected with miRNA controls or miR-130b mimics or inhibitors and luciferase reporter vectors. At 48 h posttransfection, the assay was performed to measure the luciferase activity. The relative level of luciferase activity was calculated as described above. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001.

FIG 8

gga-miR-130b inhibits the expression of SOCS5 in DF-1 cells. (A and B) Transfection of miR-130b into DF-1 cells reduced expression of SOCS5 at the protein level. DF-1 cells were transfected with miRNA controls or miR-130b mimics at 80 nM. (A) Forty-eight hours after transfection, cells were harvested, and the cytosolic proteins were examined by Western blotting using anti-SOCS5 polyclonal antibodies. GAPDH expression was used as an internal control. (B) The band densities of SOCS5 in panel A were quantitated by densitometry. The relative level of SOCS5 expression was calculated as follows: band density of SOCS5 in each sample/band density of GAPDH in the same sample. (C and D) miR-130b inhibitors enhanced the expression of SOCS5. DF-1 cells were transfected with miRNA controls or miR-130b mimics or inhibitors. (C) Forty-eight hours after transfection, the expression of SOCS5 was examined by Western blotting. (D) The band densities of SOCS5 in DF-1 cells in panel C were quantitated by densitometry, and the relative levels of SOCS5 expression were calculated as described above. Data are representative of three independent experiments and are presented as means and SD. **, P < 0.01; *, P < 0.05.

FIG 9

Inhibition of SOCS5 by gga-miR-130b enhances the expression of STATs. (A to F) DF-1 cells were transfected with miRNA controls or miR-130b mimics or inhibitors at 80 nM. Eighteen hours after transfection, cells were infected with IBDV at an MOI of 0.1 or left uninfected. Twelve hours after infection, mRNA expression levels of STAT1 (A), STAT2 (B), STAT3 (C), STAT4 (D), STAT5 (E), and STAT6 (F) were measured by qRT-PCRs using specific primers, and GAPDH was used as an internal control. The relative levels of gene expression were calculated as follows: mRNA expression of the STAT gene in miR-130b-transfected cells or normal cells/mRNA expression of the STAT gene in miRNA control-transfected cells. Data are representative of three independent experiments and are presented as means and SD. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 10

gga-miR-130b enhances STAT1 phosphorylation on Tyr701 residues. (A and B) miR-130b transfection enhanced phosphorylation of STAT1. DF-1 cells were transfected with miR-130b mimics or miRNA controls at 80 nM. Twenty-four hours after transfection, cells were stimulated with chicken IFN-γ (200 ng/ml) and harvested at the indicated time points. (A) The cell lysates were examined by Western blotting using anti-pSTAT1 (Tyr701) and anti-STAT1 antibodies. GAPDH expression was used as an internal control. (B) The band densities of p-STAT1 in panel A were quantitated by densitometry. The relative level of p-STAT1 was calculated as follows: band density of p-STAT1 in each sample/band density of GAPDH in the same sample. (C and D) miR-130b inhibitors reduced the phosphorylation of STAT1. DF-1 cells were transfected with miR-130b inhibitors or miRNA inhibitor controls and stimulated with chicken IFN-γ (200 ng/ml) for the indicated periods. (C) Phosphorylated STAT1 was examined by Western blotting. (D) The band densities of p-STAT1 in panel C were quantitated by densitometry. The relative level of p-STAT1 expression was calculated as described above. Data are representative of three independent experiments and are presented as means and SD. **, P < 0.01.