miR-Novel-80 Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Targeting the Viral Nsp1 Gene and Downregulating Host CXXC Finger Protein 4

Figures

• 

  Figure 1. miRNA screening and histopathological analysis in Tibetan and Large White pigs. (A) Schematic overview of the microRNA sequencing workflow in Tibetan and Large White pigs. (B) Histopathological examination of lung and lymph node tissues from Tibetan and Large White pigs following PRRSV infection. (C) RT-qPCR analysis of PRRSV RNA levels in lung tissues from TP-challenged, LW-challenged, TP-control, and LW-control pigs. *** p < 0.001. (D) Volcano plot of differentially expressed miRNAs in TP challenge vs. LW challenge.

• 

  Figure 2. miR-novel-80 inhibits PRRSV replication. (A,B) MARC-145 cells (A) and iPAMs (B) were transfected with the indicated miRNA mimics or negative control (NC) mimics for 10 h, followed by infection with the PRRSV JXA1 strain (MOI = 0.1) for 24 h. Viral copy numbers were subsequently determined via RT-qPCR. ** p < 0.01, **** p < 0.0001, ns: no significantly. (C) iPAMs were transfected and infected under the same conditions as in (A,B), and viral protein levels were assessed by WB analysis. (D) iPAMs were transfected with miR-novel-80 inhibitors or NC inhibitors for 10 h, followed by infection with PRRSV JXA1 (MOI = 0.1) for 24 h. PRRSV replication was evaluated using both RT-qPCR and Western blotting analyses. **** p < 0.0001. (E) A CCK-8 assay was performed on iPAMs transfected with 30, 60, or 90 nM of miR-novel-80 mimics or NC for 24 h. ns: no significantly. (F,G) iPAMs were transfected with graded doses of miR-novel-80 mimics (F) or inhibitors (G), followed by PRRSV infection for 24 h. Viral replication was subsequently analyzed by RT-qPCR and WB. ** p < 0.01, **** p < 0.0001, ns: no significantly.

• 

  Figure 3. miR-novel-80 directly targets the PRRSV type region. (A,B) iPAMs were transfected with miR-novel-80 mimics or NC and then infected with the indicated PRRSV strains [PRRSV-2 strains: SC202403, SC202401, SCwhn14XC-1 and SC202404 at an MOI of 0.05. At 24 hpi, viral RNA levels (A) and N protein expression (B) were measured to assess viral replication. miR-novel-80 inhibits the replication of multiple PRRSV genotypes and lineages. **** p < 0.0001. (C) Viral titers determined by TCID50 assays at indicated time points post-transfection with miR-novel-80. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: no significantly.

• 

  Figure 4. miR-novel-80 directly targets the conserved nsp1 gene of PRRSV. (A) Sequence alignment of the miR-novel-80 target site within the nsp1-coding region (ORF1a) of PRRSV. The target site was predicted using miRanda (v3.3a) and is highly conserved across PRRSV-1 and PRRSV-2 lineages (L1, L3, L5, and L8). (B) Schematic diagram of the predicted miR-novel-80 binding site and the corresponding mutant sequence within the PRRSV JXA1 genome. The wild-type (WT) or mutant (MUT) binding sites were cloned into the pmirGLO vector for dual-luciferase reporter assays. (C) Relative luciferase activity in 293T cells co-transfected with the dual-luciferase reporter plasmids and miR-novel-80 mimics or NC mimics for 24 h. *** p < 0.001, ns: no significantly. (D) Relative luciferase activity in 293T cells co-transfected with the dual-luciferase reporter plasmids and miR-novel-80 inhibitors or NC inhibitors for 24 h. **** p < 0.0001, ns: no significantly.

• 

  Figure 5. Transcriptomic analysis of miR-novel-80 in iPAMs. (A) Schematic overview of the experimental procedure: iPAMs were transfected with miR-novel-80 mimics or NC mimics for 24 h. Total RNA was then extracted using the TRIzol reagent, followed by high-throughput transcriptome sequencing. (B) Network visualization of potential miR-novel-80 target genes. Node size and color intensity correspond to the magnitude of differential expression. (C) Volcano plot showing the distribution of DEGs between the miR-NC and miR-novel-80 groups. The vertical dashed lines indicate the fold-change threshold (|log2FC| > 1), and the horizontal dashed line indicates the significance threshold (p < 0.05). (D) Gene ontology (GO) enrichment analysis of the identified DEGs, categorized by biological process (BP), cellular component (CC), and molecular function (MF). (E) KEGG pathway enrichment analysis of the DEGs.

• 

  Figure 6. miR-novel-80 directly targets host CXXC4. (A) RT-qPCR analysis of CXXC4, ANKS4B, Il33, NME4, and COX7A1 mRNA levels in iPAMs transfected with miR-novel-80 mimics for 24 h. ** p < 0.01, ns: no significantly. (B) WB analysis of CXXC4 protein expression in iPAMs at 24 h post-transfection with miR-novel-80 mimics. (C) Schematic representation of the predicted miR-novel-80 binding site and the corresponding mutant sequence within the 3′UTR of the CXXC4 gene. (D,E) Dual-luciferase reporter assays in 293T cells co-transfected with pmirGLO-CXXC4-WT or pmirGLO-CXXC4-MUT reporter plasmids alongside miR-novel-80 mimics (D) or miR-novel-80 inhibitors (E) for 24 h. *** p < 0.001, **** p < 0.0001, ns: no significantly.

• 

  Figure 7. miR-novel-80 inhibits PRRSV replication by targeting host CXXC4 to activate Wnt/β-catenin and NF-κB signaling. (A,B) iPAMs at 70% confluence were transfected with si-CXXC4, followed by PRRSV inoculation. PRRSV replication was assessed by RT-qPCR (A) and WB (B). *** p < 0.001. (C) WB analysis of β-catenin protein levels in iPAMs transiently transfected with miR-novel-80 mimics for 24 h. (D) RT-qPCR analysis of Wnt/β-catenin target genes (AXIN2, NKD1, c-Myc, and cyclin D1) in iPAMs transfected with miR-novel-80 mimics, si-CXXC4, or their respective negative controls at 24, 36, and 48 h post-transfection. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: no significantly. (E) Cell viability was determined using the CCK-8 assay at the indicated time points after transfection with NC or miR-novel-80 mimics. * p < 0.05, ** p < 0.01, ns: no significantly. (F) RT-qPCR analysis of IκB-α, IL-6, IL-8, TNF-α, and NF-κB mRNA levels in iPAMs transfected with si-CXXC4 for 48 h. * p < 0.05, ** p < 0.01, **** p < 0.0001. (G) WB analysis of NF-κB p65 protein levels in iPAMs transfected with si-CXXC4 for 48 h. (H) Western blot analysis of NF-κB p65 and PRRSV N protein levels in iPAMs…