The mRNA and protein of IL-8 oppositely regulate PRRSV replication via nucleoprotein and 14-3-3γ

Figures

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  Fig 1. 14-3-3γ functions as an anti-PRRSV factor. 14-3-3γ knockdown promotes PRRSV replication. MARC-145 cells (A–D), PAMs (G–J), and PK15CD163 cells (M–O) were transfected with siRNA targeting 14-3-3γ or control siRNA (NC) and then infected with TA-12. Cell samples were collected at the indicated HPI. The mRNA levels of 14-3-3γ and N were quantified by qPCR, and protein levels were detected by Western blotting. Viral titers in the supernatant were measured. Overexpression of 14-3-3γ inhibits PRRSV replication. MARC-145 cells (E and F), PAMs (K and L), and PK15CD163 cells (P and Q) were transfected with 3×Flag-14-3-3γ or an empty vector and then infected with TA-12. Cell samples were collected at the indicated HPI. Total RNA and protein were extracted and analyzed by qPCR and Western blotting, respectively. Viral titers in the supernatant were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for qPCR, and β-actin was used as an internal control for Western blotting. Error bars represent the SD of three experimental replicates. Asterisks indicate significant differences compared to controls: *P < 0.05; P < 0.01 (unpaired two-tailed Student’s…

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  Fig 2. 14-3-3γ downregulates PRRSV infection-induced IL-8 mRNA levels. (A) Identification of the 14-3-3γ-knockdown cell line, MARC-145si14-3-3γ, by qPCR and Western blotting. (B) Cell growth kinetics of MARC-145siNC and MARC-145si14-3-3γ. Cells were plated in 24-well plates, and at 12, 18, 24, 36, 48, 72, and 96 h post-plating, the Cell Counting Kit-8 reagent was added. After a 3 h incubation at 37°C, cell counting was performed by measuring absorbance at 450 nm. (C–H) Effect of 14-3-3γ on the expression of PRRSV-induced cytokines. TA-12 was inoculated into MARC-145siNC and MARC-145si14-3-3γ cells. Cell samples were collected at 0, 4, 8, 12, 18, and 24 HPI, and total RNA was extracted. The mRNA levels of IL-1β (C), IL-10 (D), IL-8 (E), TNF-α (F), IFN-α (G), and IFN-γ (H) were measured by relative qPCR. (I) MARC-145 cells were transfected with 3×Flag-14-3-3γ or an empty vector and then infected with TA-12. Cell samples were collected at 24 HPI, and IL-8 mRNA levels were measured by relative qPCR. (J, L) 14-3-3γ knockdown upregulates IL-8 mRNA. PAMs (J) and PK15CD163 cells (L) were transfected with siRNA targeting 14-3-3γ or non-targeting control (NC) for 24 h and then infected with…

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  Fig 3. IL-8 mRNA positively correlates with PRRSV replication. (A–D) PDTC decreases both IL-8 mRNA levels and viral genome replication. MARC-145siNC and MARC-145si14-3-3γ cells were pretreated with 0, 300, 500, 800, and 1,000 µM PDTC for 1 h and then inoculated with TA-12. Cell samples were collected at 24 HPI, and the mRNA levels of IL-8 (A) and the viral genome (B) were detected by qPCR. PAM cells were transfected with siRNA targeting 14-3-3γ or control siRNA (NC) and then pretreated with 300 µM PDTC for 1 h. Cell samples were collected after infection with TA-12 at 0, 6, 12, 24, and 36 HPI. The mRNA levels of IL-8 (C) and the viral genome (D) were detected by qPCR. (E–I) Knockdown of IL-8 inhibits PRRSV replication. MARC-145 cells (E, G, H) and PAMs (F, G, I) were transfected with siRNA targeting IL-8 or control siRNA (NC) and then infected with TA-12. Cell samples were collected at the indicated HPI. TA-12 infection was evaluated based on the N gene by qPCR, Western blotting, and viral titration. (J–M) PRRSV replication is closely related to IL-8 mRNA and protein levels. MARC-145 cells were inoculated with TA-12 for 2 h and then transfected with 3×Flag-IL-8 or an empty vector. Cell…

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  Fig 4. IL-8 mRNA positively correlates with PRRSV replication independent of its open reading frame. (A) Sequence of mutant IL-8 mRNA. (B–D) Mutant IL-8 mRNA positively correlates with PRRSV replication (method 1). MARC-145 cells were infected with TA-12 for 2 h and then transfected with 3×Flag-IL-8, 3×Flag-IL-8 (TGA), 3×Flag-IL-8 (GCA), or an empty vector. Cell samples were collected at the indicated HPI. The TA-12 genome was detected by qPCR (B), the level of IL-8 mRNA was measured by qPCR (C), and viral protein levels were assessed by Western blotting (D). (E–H) Mutant IL-8 mRNA positively correlates with PRRSV replication (method 2). MARC-145 cells were transfected with 3×Flag-IL-8, 3×Flag-IL-8 (TGA), 3×Flag-IL-8 (GCA), or an empty vector for 12 h and then inoculated with TA-12. Cells were collected at the indicated HPI. The level of IL-8 mRNA was measured by qPCR (E), the TA-12 genome was detected by qPCR (F), viral titers were determined (G), and viral protein levels were assessed by Western blotting (H). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for qPCR. Error bars represent the SD of three experimental replicates. GAPDH was used as an…

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  Fig 5. IL-8 protein inhibits PRRSV infection. (A–E) IL-8 transfection inhibits PRRSV replication. MARC-145 cells and PAMs were transfected with 3×Flag-IL-8 or an empty vector for 24 h and then infected with TA-12. Cell samples were collected at 6, 12, 24, and 36 HPI. The TA-12 genome was detected by qPCR (A, C), viral protein levels were measured by Western blotting (B), and viral titers were determined (D, E). (F, G) Cytotoxicity of IL-8 protein. The cytotoxicity of IL-8 protein was assessed using the Cell Counting Kit-8 (CCK-8) assay. Monolayers of MARC-145 cells (F) and PAMs (G) were treated with IL-8 protein at different concentrations for 24 h, followed by the addition of the CCK-8 reagent. Cell viability was measured by absorbance at 450 nm. (H–L) IL-8 protein inhibits TA-12 replication. MARC-145 cells and PAMs were inoculated with TA-12 for 2 h, and IL-8 protein at different concentrations was added. Cell samples were collected at 24 HPI. The TA-12 genome was detected by qPCR (H, J), viral titers were determined (I, K), and viral protein levels were measured by Western blotting (L). (M–Y) IL-8 protein inhibits replication of different PRRSV strains. MARC-145 cells and PAMs were…

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  Fig 6. N protein upregulates IL-8 transcription dependent on its nuclear localization. (A) Viral proteins regulate IL-8 transcription. Constructs encoding viral proteins and pGL3-IL-8-Luc (containing the IL-8 promoter) were co-transfected into HEK293T cells for 24 h. Cells were collected, and firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay. (B) N protein activates IL-8 transcription. After co-transfection of NSP2, NSP3, NSP4, and N with pGL3-IL-8-Luc into MARC-145 cells, cells were collected, and firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay. (C) Schematic structure of truncated and mutant N proteins. (D–F) Identification of key sites in N regulating IL-8 transcription. Truncated mutants of N were co-transfected with pGL3-IL-8-Luc into 293T cells, and cells were collected for dual-luciferase reporter assays. (G) Role of N proteins from different PRRSV strains in regulating IL-8 transcription. N proteins from various PRRSV strains were co-transfected with pGL3-IL-8-Luc into 293T cells, and cells were collected for dual-luciferase reporter assays after 24 h. (H) Schematic diagrams of mutant N…

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  Fig 7. 14-3-3γ and IL-8 competitively bind to N protein to modulate its nuclear distribution. (A–C) 14-3-3γ modulates the nuclear distribution of N. MARC-145 cells were transfected with Myc-14-3-3γ (A) or si14-3-3γ (B) for 24 h and then infected with TA-12. Cell samples were collected at 30 HPI, and cytoplasmic and nuclear proteins were extracted and analyzed by Western blotting. MARC-145siNC and MARC-145si14-3-3γ cells were infected with TA-12 for 30 h, and cytoplasmic and nuclear proteins were extracted and analyzed by Western blotting (C). (D, E) IL-8 protein modulates the nuclear distribution of N. MARC-145 cells were transfected with Myc-IL-8 (D) or siIL-8 (E) for 24 h and then infected with TA-12. Cell samples were collected at 30 HPI, and cytoplasmic and nuclear proteins were extracted and analyzed by Western blotting. (F) Interaction between N and 14-3-3γ. MARC-145 cells were either mock-infected or infected with HP-PRRSV TA-12 at an MOI of 0.01. Twenty-four hours later, cell lysates were pre-cleared with blank protein G resin and incubated with antibodies specific for 14-3-3γ. Proteins were analyzed by Western blotting. (G) Competitive interaction between N, 14-3-3γ, and IL-8.…