miR-191-5p suppresses PRRSV replication by targeting porcine EGFR to enhance interferon signaling

Figure 1. miR-191-5p inhibits PRRSV infection. (A,B) miR-191-5p mimic represses PRRSV infection. Primary PAMs were transfected with the mimic of miR-191-5p, miR-novel-029 or negative control (NC) at the concentration of 100 nM for 24 h, followed by inoculation with HuN4 at MOI of 0.1 for an additional 24 h. Cell lysates were subjected to western blot to detect PRRSV-N protein expression (A). Relative viral RNA level was determined by qPCR (B). (C) Transfection of miRNAs’ mimic does not affect cell viability. A CCK8 assay was performed on PAMs transfected with the indicated miRNAs mimics or NC for 72 h. (D,E) miR-191-5p inhibitor promotes PRRSV infection. PAMs were transfected with miRNA inhibitor or inhibitor-NC for 24 h, and cells were then inoculated with HuN4 at MOI of 0.1 for 24 h. Cell lysates were subjected to western blot to detect PRRSV-N protein expression (D). Relative viral RNA level was quantified by qPCR (E). (F) Transfection of miRNAs’ inhibitor does not affect cell viability. A CCK8 assay was performed on PAMs transfected with indicated miRNAs inhibitors or NC for 72 h. Data are presented as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 2. miR-191-5p Inhibits PRRSV replication. (A,B) miR-191-5p mimic represses PRRSV infection. PAMs were transfected with miR-191-5p mimic or miR-NC for 24 h, and the cell monolayers were then inoculated with HuN4 at MOI of 0.1 for 24 h. Cells supernatants were collected for detection of viral titers (A). Virus infection was examined by IFA (B). (C) miR-191-5p mimic dose-dependently inhibits PRRSV. PRRSV-N protein was detected by transfection with different miR-191-5p mimic concentrations. (D,E) miR-191-5p inhibitor promotes PRRSV infection. PAMs were transfected with miR-191-5p inhibitor or inhibitor-NC for 24 h, followed by inoculation with HuN4 at MOI of 0.1 for 24 h. Cells supernatants were collected for determined viral titer (D). (E) Virus infection was examined by IFA. (F) miR-191-5p inhibitor promotes PRRSV. PRRSV-N protein was detected by transfection with different concentrations of miR-191-5p inhibitor. (G) Overexpression of miR-191-5p decreases PRRSV replication. Transfection of miR-191-5p was followed by inoculation of HuN4 with MOI of 0.1, and then cells were collected at different time points. The relative level of viral RNA was determined by qPCR. Data are presented as…

Figure 3. miR-191-5p negatively regulates the expression of porcine EGFR. (A–C) miR-191-5p mimic represses PRRSV infection in PAMs but not MARC-145 or HEK293-CD163. After transfection of miR-191-5p or miR-NC mimic into PAM (A), HEK293-CD163 (B) and MARC-145 (C) for 24 h, and the cells were then infected with PRRSV at MOI of 0.1 for 24 h. Cells lysates were subjected to western blot to detect the expression of PRRSV-N protein. Relative viral RNA level was determined by qPCR. (D) Wayne plots of miR-191-5p 3’UTR predicted target in pig, human and monkey. (E) EGFR was downregulated by miR-191-5p. PAMs were transfected with miR-191-5p or miR-NC for 24 h. Total RNA was extracted, and the mRNA level were determined by qPCR. (F) diagram of the predicted miR-191-5p binding sites in EGFR 3’UTR of different species. miR-191-5p seed region and its predicted binding sites are indicated in bold. Bases with mutations and differences in seed prediction binding regions are underlined. (G) miR-191-5p directly targets EGFR 3’UTR. HEK293T cells were co-transfected with EGFR 3’UTR wild type or mutant luciferase reporter vector (100 ng), along with miR-191-5p mimics or NC for 48 h and then harvested for…

Figure 4. Inhibition of Endogenous EGFR suppresses PRRSV replication. (A,B) EGFR promotes PRRSV infection. PAMs were incubated with EGFR lentivirus mixed with polybrene for 12 h and then infected with PRRSV for 24 h. Cells were collected to detect PRRSV RNA and protein separately. (C–E) EGFR facilitates PRRSV infection. PAMs were stimulated with different concentrations of EGF and then inoculated with PRRSV for 24 h. Cells were collected to detect PRRSV RNA and protein separately. (F,G) EGF and gefitinib have no effect on PAMs’s viability. CCK8 assay was performed on PAMs treated with EGF or gefitinib for 72 h at indicated concentration. (H) Inhibition of EGFR phosphorylation by gefitinib in PAMs. After 24 h of gefitinib pretreatment, PAMs were stimulated with EGF for 30 min and then harvested for western blot detection of protein expression. (I,J) Gefitinib inhibits PRRSV infection. PAMs were treated with gefitinib at a concentration of 7 μM for 24 h, the cells were then inoculated with PRRSV for another 24 h. The cells were collected to detect PRRSV-N protein (I) and RNA (J). (K) Verification of EGFR knocking down. PAMs were transfected with EGFR-specific siRNA (si-EGFR) or si-NC for 24…

Figure 5. miR-191-5p regulates viral replication by modulating interferon activity through EGFR. (A) EGFR inhibit IFN-β promoter luciferase activity. The IFN-β promoter and pRL-TK were co-transfected into HEK293T cells along with EGFR, IRF3, and control, respectively. The cells were collected for luciferase activity assay after 48 h. (B) PAMs were inoculated with 20 ng/mL of EGF for 24 h, and the cells were collected for qPCR to detect the RNA level of the indicated gene. (C,D) PAMs were transfected with miR-191-5p or si-EGFR for 24 h. The RNA level was determined by qPCR. (E) PAMs were transfected with miR-191-5p and after 24 h the cells were treated with EGF. Total RNA was extracted, and the mRNA levels of IFN-β, were determined by qPCR. (F) PAMs were treated with EGF, then cells were infected with PRRSV for 24 h. Total RNA was extracted to detect the expression levels of PRRSV RNA. Data are presented as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 6. STAT3 promotes PRRSV replication in PAMs. (A,B) The effect of STAT3 inhibitor on PRRSV. PAMs were pretreated with 60 μM S3I-201 for 24 h and then inoculated with PRRSV at 0.1 MOI for 24 h. Cells were collected to detect the expression levels of PRRSV RNA (A) and protein (B). (C) The effect of S3I-201 on cell viability. CCK8 assay was performed on PAMs treated with S3I-201 for 72 h at the indicated concentration. (D) PAMs were pretreatment with S3I-201 for 24 h and stimulated with EGF for 30 min. Cells were then harvested for western blot to detect the indicated protein expression. (E) Verification of si-STAT3 knockdown efficiency. The mRNA level of STAT3 was detected in PAMs after transfection of si-STAT3 for 24 h. (F,G) Effect of knocking down endogenous STAT3 on PRRSV. PAMs were transfected with si-STAT3 for 24 h and then infected with PRRSV for 24 h. Cells were collected to detect the expression levels of PRRSV RNA (F) and protein (G). (H,I) STAT3 promotes PRRSV infection. PAMs were incubated with STAT3 lentivirus mixed with polybrene for 12 h and then infected with PRRSV for 24 h. Cells were collected to detect PRRSV RNA (H) and protein (I) separately. Data are presented as…

Figure 7. STAT3 is downstream of EGFR in regulating interferon signaling during EGF activation in PAMs. (A) STAT3 inhibits IFN-β promoter luciferase activity. The IFN-β promoter and pRL-TK were co-transfected into HEK293T along with STAT3, IRF3, and control, respectively. The cells were collected for luciferase activity assay after 48 h. (B,C) PAMs were treated with the inhibitor S3I-201 or transfected with si-STAT3 for 24 h. RNA was extracted to detect the expression levels of indicated mRNA using qPCR. (D) Immortalized PAM were co-transfected with the IFN-β promoter and pRL-TK along with si-STAT3 or si-NC for 12 h, and then treated with EGF or control for another 24 h. The cells were collected for luciferase activity assay. (E) PAMs were transfected with si-STAT3 for 12 h, followed by treatment with EGF for 24 h, and the cells were collected for detection of IFN-β mRNA level. (F) PAMs were treated with EGF, then cells were infected with PRRSV for 24 h. Total RNA was extracted to detect the expression levels of PRRSV RNA. (G) PAMs were treated with S3I-201 for 12 h, then transfected with miR-191-5p inhibitor or NC for 12 h, followed by infection with PRRSV for 24 h. The cells were…

Figure 8. miR-191-5p broadly inhibits various PRRSV strains. (A–C) miR-191-5p overexpression decreases CH-1a, JXA1, and MLV replication. PAMs were transfected with miR-191-5p or miR-NC for 24 h and then infected with the indicated PRRSV strains at 0.1 or 0.01 MOI for another 24 h. Cells were collected, and PRRSV RNA and protein were detected separately. Data are presented as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01.