miR-541-3p Promoted Porcine Reproductive and Respiratory Syndrome Virus 2 (PRRSV-2) Replication by Targeting Interferon Regulatory Factor 7

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by PRRS virus (PRRSV), which seriously harms the pig industry. Revealing the mechanism by which PRRSV inhibits immune response will help prevent and control PRRS. Here, we found that PRRSV-2 may hijack host miR-541-3p to inhibit host innate immune response. Firstly, this work showed that miR-541-3p mimics could facilitate the replication of PRRSV-2 and the results of the quantitative real time polymerase chain reaction (qRT-PCR) showed that PRRSV-2 could up-regulate the expression of miR-541-3p in MARC-145 cells. Since previous studies have shown that type I interferon could effectively inhibit the replication of PRRSV-2, the present work explored whether miR-541-3p regulated the expression of type I interferon and found that miR-541-3p could negatively regulate the transcription of type I interferon by targeting interferon regulatory factor 7 (IRF7). More importantly, PRRSV-2 infection could down-regulate the expression of IRF7 and over-expression of IRF7 could down-regulate the replication of PRRSV-2 in MARC-145 cells. In conclusion, PRRSV-2 infection up-regulated the expression of miR-541-3p to promote its replication in MARC-145 cells, since miR-541-3p can negatively regulate the transcription of type I interferon by targeting IRF7.

Keywords: IRF7; PRRSV; miR-541-3p; type I interferon.

Figure 1

miR-541-3p promoted the replication of PRRSV-2 in MARC-145 cells. (A) MARC-145 cells were transfected with miR-379-5p mimics, miR-541-3p mimics, miR-491-5p mimics, miR-329-3p mimics, miR-1283 mimics or miR-449b-3p mimics, and 24 h later, the cells were infected with PRRSV-2 at a multiplicity of infection (MOI) of 0.1. Twenty-four hours later, the cells were collected for the analysis of RNA levels of Open reading frame 7 (ORF7) of PRRSV-2 by quantitative real time polymerase chain reaction (qRT-PCR). MARC-145 cells were transfected with miR-382-5p mimics (B,C), miR-382-5p inhibitors (D,E), negative control mimics (NC-mimics) or negative control inhibitors (NC-inhibitors) at a final concentration of 50 nM or 100 nM, and 24 h later, the cells were infected with PRRSV at an MOI of 0.1. Twenty-four hours later, the cells were harvested for the analysis of RNA levels of PRRSV-2 by qRT-PCR (B,D) and for the analysis of the PRRSV-2 titers by 50% tissue culture infected dose (TCID₅₀) (C,E). Data are presented as the mean + standard deviations (SD). Student’s t test was used for statistical analysis. All experiments were repeated at least three times with similar results. ** p < 0.01.

Figure 2

PRRSV-2 infection up-regulated the expression of miR-541-3p in MARC-145 cells. (A) MARC-145 cells were infected with PRRSV-2 (multiplicity of infection (MOI) = 1) for indicated times. Then, the expression levels of miR-541-3p were determined by quantitative real time polymerase chain reaction (qRT-PCR). (B) MARC-145 cells were infected with PRRSV-2 at different MOIs, and 24 h later, the expression levels of miR-541-3p were measured by qRT-PCR. All experiments were repeated at least three times with similar results. ** p < 0.01.

Figure 3

miR-541-3p inhibited poly(I:C)-induced production of type I interferon. MARC-145 cells were transfected with p-284, pRL-TK and miR-541-3p mimics (A), miR-541-3p inhibitors (C) or pcDNA6.2-miR-541-3p (E), and 36 h later, the cells were transfected with poly(I:C) (10 µg/mL), and 9 h later, the IFN-β luciferase activities were assayed by using the dual-luciferase reporter assay. MARC-145 cells were transfected with miR-541-3p mimics (B), miR-541-3p inhibitors (D) or pcDNA6.2-miR-541-3p (F), and 36 h later, the cells were transfected with poly(I:C) (10 µg/mL), and 9 h later, the mRNA expression levels of IFN-β were assayed by quantitative real time polymerase chain reaction (qRT-PCR). Ctrl indicated that the cells were not transfected with poly(I:C). All experiments were repeated at least three times with similar results. ** p < 0.01.

Figure 4

Interferon regulatory factor 7 (IRF7) was a target gene of miR-541-3p. HEK 293T cells were transfected with miR-541-3p mimics (A), miR-541-3p inhibitors (B), NC-mimics or NC-inhibitors. Then, 48 h later, the expression of predicted target mRNAs of miR-541-3p was measured by quantitative real time polymerase chain reaction (qRT-PCR). HEK 293T cells were transfected with miR-541-3p mimics at a final concentration of 50 nM or 100 nM (C), miR-541-3p inhibitors at a final concentration of 50 nM or 100 nM (D) and NC-mimics or NC-inhibitors. Then, 48 h later, the expression levels of IRF7 were measured by qRT-PCR. (E) Schematic presentation of base pairing between the 3′ untranslated region (UTR) of IRF-7 and the miR-541-3p sequence; the underlined red bases are their paired bases and their corresponding mutant bases. HEK 293T cells were transfected with the pcDNA6.2-miR-541-3p or pcDNA6.2-Mut-miR-541-3p and psiCHECK-2-IRF7-3′-UTR or psiCHECK-2-Mut-IRF7-3′-UTR, and 24 h later, the cells were harvested for luciferase activity assay by using the dual-luciferase reporter assay system. All experiments were repeated at least three times with similar results. * p < 0.05; ** p < 0.01.

Figure 5

PRRSV-2 infection down-regulated the expression of interferon regulatory factor 7 (IRF7). (A) MARC-145 cells were infected with PRRSV-2 at a multiplicity of infection (MOI) of 0.1, and 36 h later, the mRNA expression levels of IRF7 were assessed by quantitative real time polymerase chain reaction (qRT-PCR). (B) MARC-145 cells were infected with PRRSV-2 at different MOI, and 24 h later, the protein expression levels of IRF7 were assessed by Western blots. (C) MARC-145 cells were infected with PRRSV-2 at an MOI of 0.1, and 24 h, 36 h or 48 h later, the protein expression levels of IRF7 were assessed by Western blots. All experiments were repeated at least three times with similar results. ** p < 0.01.

Figure 6

Interferon regulatory factor 7 (IRF7) inhibited the replication of PRRSV-2. MARC-145 cells were transfected with siIRF7 or siNC, and 48 h later, the protein levels (A) and the mRNA expression levels (B) of IRF7 were assessed by Western blots and quantitative real time polymerase chain reaction (qRT-PCR), respectively. (C) MARC-145 cells were transfected with siIRF7, and 24 h later, the cells were infected with PRRSV-2, and then the cells were harvested and the RNA levels of Open reading frame 7 (ORF7) of PRRSV-2 were determined by qRT-PCR. (D) MARC-145 cells were transfected with pCMV-IRF7-Flag, and 24 h later, the protein expression levels of IRF7 were assessed by Western blots. (E) MARC-145 cells were transfected with pCMV-IRF7-Flag, and 24 h later, the cells were infected with PRRSV-2, and then the cells were harvested and the RNA levels of PRRSV-2 ORF7 were determined by qRT-PCR. All experiments were repeated at least three times with similar results. ** p < 0.01.