MicroRNA let-7f-5p Inhibits Porcine Reproductive and Respiratory Syndrome Virus by Targeting MYH9

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Current antiviral strategies do not effectively prevent and control PRRSV. Recent reports show that microRNAs (miRNAs) play vital roles in viral infections by post transcriptionally regulating the expression of viral or host genes. Our previous research showed that non-muscle myosin heavy chain 9 (MYH9) is an essential factor for PRRSV infection. Using bioinformatic prediction and experimental verification, we demonstrate that MYH9 expression is regulated by the miRNA let-7f-5p, which binds to the MYH9 mRNA 3'UTR and may play an important role during PRRSV infection. To understand how let-7f-5p regulates PRRSV infection, we analyzed the expression pattern of both let-7f-5p and MYH9 in porcine alveolar macrophages (PAMs) after infection with either highly pathogenic PRRSV (HP-PRRSV) or classical type PRRSV (N-PRRSV) using a deep sequencing approach with quantitative real-time PCR validation. Our results showed that both HP-PRRSV and N-PRRSV infection reduced let-7f-5p expression while also inducing MYH9 expression. Furthermore, let-7f-5p significantly inhibited PRRSV replication through suppression of MYH9 expression. These findings not only provide new insights into the pathogenesis of PRRSV, but also suggest potential new antiviral strategies against PRRSV infection.

Figure 1. Luciferase reporter activity assay identifies let-7f-5p as a regulator of MYH9.

(a) Pig MYH9 3′UTR and monkey MYH9 3′UTR were cloned into 3′UTR-luciferase reporter psiCheck2 vectors to create plasmids designated psiCheck2-pMYH9-WT and psiCheck2-mMYH9-WT, respectively. (b) HEK293FT cells were cotransfected with indicated mimics and psiCheck2-pMYH9-WT. (c) HEK293FT cells were cotransfected with indicated mimics and psiCheck2-mMYH9-WT. Thirty-six hrs later, cells were lysed and luciferase activity was measured. Results are expressed as mean ± SD of three independent experiments. p values were calculated using the Student’s t test. **P < 0.01, ***P < 0.001. (d) Sequence homology of let-7f-5p to human, monkey, and pig.

Figure 2. Let-7f-5p specifically binds to the 3′ UTR of pig MYH9.

(a) The 3′UTR of pig MYH9 contains one seed-matched target site (highlighted) for let-7f-5p. (b) Schematic of the “seed region” match between let-7f-5p and pig MYH9 3′UTR. The mutation of six nucleotides in the seed match is shown. The positions of six seed match sites for let-7f-5p in the pig MYH9 3′UTR were replaced. (c) WT or MUT reporter plasmids were cotransfected with the let-7f-5p mimics, let-7f-5p-MUT mimics, or NC mimics into the HEK293FT cells. Reporter activities were determined thirty-six hrs post-transfection by dual-luciferase assays. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student’s t test. ***P < 0.001. (d) Sequence homology of the let-7f-5p binding sequence in the MYH9 3′UTR of human, monkey, and pig.

Figure 3. Let-7f-5p specifically binds to the 3′ UTR of monkey MYH9.

(a) The 3′UTR of monkey MYH9 contains one seed matched target site (highlighted) for let-7f-5p. (b) Schematic of the “seed region” match between let-7f-5p and monkey MYH9 3′UTR. The mutation of six nucleotides in the seed match is shown. The positions of six seed match sites for let-7f-5p in the monkey MYH9 3′UTR were replaced. (c) WT or MUT reporter plasmids were cotransfected with the let-7f-5p mimics, let-7f-5p-MUT mimics, or NC mimics into HEK293FT cells. Reporter activities were determined thirty-six hrs post-transfection by dual-luciferase assays. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student’s t test. ***P < 0.001.

Figure 4. Let-7f-5p physically binds to MYH9 mRNA in the RISC.

RNA of mock- and GD-HD-infected PAMs were subjected to Ago2-IP, the relative expression of let-7f-5p (a) and MYH9 mRNA (b) in the immunoprecipitates were determined by qRT-PCR. As a negative control, immunoprecipitation was performed using anti-Flag antibody beads (IgG). Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student’s t test. *P < 0.05, ***P < 0.001.

Figure 5. Overexpression of let-7f-5p decreases MYH9 mRNA and protein levels in PAMs.

PAMs were transfected with 100nM of MYH9-siRNA, NC-siRNA, let-7f-5p mimics, and NC-mimics. Relative expression level of let-7f-5p (a) and MYH9 mRNA (b) in PAMs determined by qRT-PCR. MYH9 protein expression (c) analyzed by western blot with α-tubulin as the control. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student’s t test. **P < 0.01, ***P < 0.001.

Figure 6. Inhibition of let-7f-5p enhances MYH9 mRNA and protein expression in PAMs.

PAMs were transfected with indicated concentrations of MYH9-siRNA, NC-siRNA, let-7f-5p-inhibitor, and NC-inhibitor. Relative expression level of let-7f-5p (a) and MYH9 mRNA (b) in PAMs determined by qRT-PCR. MYH9 protein expression (c) analyzed by western blot, with α-tubulin as control. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student’s t test. *P < 0.05, **P < 0.01.

Figure 7. Let-7f-5p is reduced after PRRSV infection.

(a) Heatmap for let-7 family members at 12 hpi compared with control group in the PAMs infected with two PRRSV strains, GD-HD and CH-1a (MOI = 0.1), quantified by microRNA deep sequencing. The log2 fold changes were used to plot the heatmap. PAMs cells were mock-infected or were infected with UV-inactivated or active forms of two PRRSV strains, GD-HD and CH-1a (MOI = 0.1). PRRSV ORF7 (b,c), let-7f-5p (d,e), and MYH9 (f,g) mRNA relative expression levels were determined using qRT-PCR. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8. Characterization of the anti-PRRSV activity of let-7f-5p.

PAMs were transfected with NC mimics or various concentrations of let-7f-5p and infected with PRRSV strain GD-HD (MOI = 0.01) for 24 hrs. PRRSV replication was evaluated by qRT-PCR to measure levels of extracellular PRRSV genome RNA in the supernatants (a), intracellular PRRSV ORF7 RNA (b), and western blot for intracellular PRRSV N protein with α-tubulin as control (c). Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student’s t test. **P < 0.01, ***P < 0.001.

Figure 9. Overexpression of let-7f-5p attenuates both HP-PRRSV and N-PRRSV replication in PAMs.

PAMs were transfected with 100 nM let-7f-5p or NC mimics, followed by infection with two PRRSV strains: GD-HD and CH-1a (MOI = 0.01). (a,b) Relative expression level of MYH9 mRNA was evaluated by qRT-PCR. PRRSV replication was evaluated by qRT-PCR for intracellular PRRSV ORF7 RNA (c,d) and extracellular PRRSV genome RNA in the supernatants (e,f), and viral titers in the supernatants were measured by TCID₅₀ at the indicated time points (g,h). Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.