The C1QTNF6–MX2 Antiviral Axis Inhibits Porcine Circovirus Type 2 Replication in Porcine Macrophages

Figures

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  Figure 1. C1QTNF6 expression characteristics. (A) C1QTNF6 expression across various pig tissues at different stages of gestation. n = 3. Gd25: Gestation day 25, Gd40: Gestation Day 40, Gd80: Gestation Day 80, Gd112: Gestation Day 112. (B) Analysis of C1QTNF6 protein expression profile based on protein profiles of human tissues.

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  Figure 2. C1QTNF6 significantly modulates the proliferative activity of 3D4/21 cells. (A) RT-PCR (n = 3) and Western blotting detected the efficiency of C1QTNF6 overexpression, n = 2. (B,C) Flow cytometry analysis to detect apoptosis in 3D4/21 cells after C1QTNF6 overexpression, n = 3. (D) RT-PCR to detect expression changes in cycle-related genes after C1QTNF6 overexpression. (E,F) Western blotting analysis the expression of apoptosis-related protein (Bax and Bcl2), n = 2. Statistical analyses were performed by using unpaired two-tailed Student’s t-test. ns: No significantly. * p < 0.05, ** p < 0.01.

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  Figure 3. C1QTNF6 significantly promotes the expression of immune-related genes. (A) RT-PCR to detect changes in inflammatory factor-related gene expression after C1QTNF6 overexpression. n = 3. (B) RT-PCR to detect changes in interferon-related gene expression after C1QTNF6 overexpression, n = 3. Statistical analyses were performed by using unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01.

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  Figure 4. C1QTNF6 overexpression upregulates immune-related gene expression in 3D4/21 cells. (A) PCA group clustering analysis after C1QTNF6 overexpression. (B) Gene volcano map after C1QTNF6 overexpression. (C) Heat map of 68 genes after C1QTNF6 overexpression. FC ≥ 0.5 and p-adj < 0.05. (D) Functional enrichment analysis results of up-regulated expressed genes after C1QTNF6 overexpression, only Top10 entries are shown. (E) RT-PCR validation of differentially expressed genes screened by RNA-Seq, n = 3. (F) IGV showing representative genes with altered mRNA expression levels after C1QTNF6 overexpression.

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  Figure 5. PCV2 infection markedly downregulates C1QTNF6 expression in 3D4/21 cells. (A) Expression analysis of CAP genes (n = 3) and protein at different time points in PCV2-infected 3D4/21 cells. Statistical analyses were performed by multiple t-test. (B) Alterations in C1qtnf6-regulated gene expression following 48 h PCV2 infection in 3D4/21 cells, n = 3. (C) Alterations in inflammatory factor-related genes expression following 48 h PCV2 infection in 3D4/21 cells, n = 3. (D) Alterations in interferon-related genes expression following 48 h PCV2 infection in 3D4/21 cells, n = 3. (E) Expression changes in C1qtnf6 following 48 h PCV2 infection in 3D4/21 cells, n = 3. (F) Changes in the expression of ISG15 protein after PCV2 infection of cells for 48h, n = 3. Statistical analyses were performed by using unpaired two-tailed Student’s t-test. ns: No significantly. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 6. C1QTNF6 inhibited PCV2 virus replication by upregulating the MX2 gene. (A,B) Expression of PCV2 Cap gene and protein in C1QTNF6 overexpressing 3D4/21 cells after PCV2 infection, n = 3. (C) Differential expression of C1QTNF6 regulated genes at 48 h post infection in C1QTNF6 overexpressing cells, n = 3. (D) Inflammatory cytokine expression in PCV2 infected 3D4/21 cells overexpressing C1QTNF6, n = 3. (E) Interferon related gene expression in PCV2 infected 3D4/21 cells overexpressing C1QTNF6, n = 3. (F) Validation of MX2 overexpression efficiency by RT qPCR and Western blotting, n = 3. (G) MX2 expression in MX2 overexpressing 3D4/21 cells at after PCV2 infection, n = 3. (H–J) PCV2 Cap mRNA and protein expression in MX2-overexpressing 3D4/21 cells at 48 h post-infection, n = 3. Statistical analyses were performed by using unpaired two-tailed Student’s t-test. ns: No significantly. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 7. Mechanism of C1QTNF6 inhibiting PCV2 replication in association with MX2 upregulation.