EGCG Restricts PRRSV Proliferation by Disturbing Lipid Metabolism

FIG 1. Effects of EGCG on cell viability, cell cycle, and apoptosis. (A and B) MARC-145 (A) and PAM (B) cells were treated with EGCG (0–10 μM) for 12–72 h. Cell viability was assessed with CCK-8 assay. h.p.t, hours posttreatment. (C and D) MARC-145 (C) and PAM (D) cells were treated with EGCG (10 μM) for 0–36 h. The cell cycle was assessed with Hoechst 33342 staining in flow cytometry. (E–G) MARC-145 and PAM cells were treated with EGCG (0–10μM) for 36 h. Apoptosis was assessed with Annexin V-FITC and PI staining in flow cytometry (E). Quantification of the percentage of cell death was shown in F and G.

FIG 2. EGCG inhibited PRRSV proliferation in vitro. (A) MARC-145 cells were infected with PRRSV-GFP (MOI = 10) and treated with EGCG (0–10 μM) for 48 h. Viral replication was analyzed by fluorescence microscopy. Scale bar, 100 μm. (B) GFP-positive cells from A were analyzed by flow cytometry. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) MARC-145 cells were treated as in A. The mRNA level of PRRSV ORF7 was assessed by qRT-PCR analysis. *, P < 0.05; ***, P < 0.001. (D and E) MARC-145 (D) and PAM (E) cells were treated as in A. PRRSV-N and GAPDH were assessed by immunoblotting analysis. (F and G) MARC-145 and PAM cells were infected with LP-PRRSV (F, MOI = 10) and HP-PRRSV (G, MOI = 10), and treated with EGCG (0–10 μM) for 48 h. Viral titers were assessed by TCID50 assay.

FIG 3. EGCG impaired PRRSV replication and assembly. (A) HP-PRRSV (MOI = 10) was allowed to bind to the surfaces of MARC-145 cells in the absence or presence of EGCG (10 μM) on ice for 1 h. After the cells were washed three times with ice-cold PBS, total RNA was extracted and reverse-transcribed to cDNA. Viral attachment was assessed with qRT-PCR analysis of PRRSV ORF7 mRNA. ns, no significance. (B) HP-PRRSV (MOI = 10) was allowed to bind to the surfaces of MARC-145 cells on ice for 1 h, and the cells were then shifted to 37°C in the absence or presence of EGCG (10 μM) for 2 h. After the cells were washed three times with PBS, total RNA was extracted and reverse-transcribed to cDNA. Viral entry was assessed with the qRT-PCR analysis of PRRSV ORF7 mRNA. ns, no significance. (C) MARC-145 cells were infected with HP-PRRSV (MOI = 10) and treated with EGCG (10 μM) for 6–48 h. Viral replication was assessed with dsRNA staining. Scale bar, 100 μm. (D) Quantification of the relative fluorescence intensities of dsRNA from C. ***, P < 0.001. (E) MARC-145 cells were infected with HP-PRRSV (MOI = 10) and treated with EGCG (10 μM) for 24 h. The efficiency of viral assembly in the supernatants was…

FIG 4. EGCG reduced lipid droplet formation and lipid content. (A) MARC-145 and PAM cells were infected with HP-PRRSV (MOI = 10) and treated with EGCG (10 μM) for 0–36 h. Lipid droplets were detected by Oil Red O staining. Scale bar, 10 μm. Quantifications of lipid droplets numbers per cell are shown on the right. ***, P < 0.001. ns, no significance. (B) MARC-145 and PAM cells were treated as in A. Lipid droplets were detected by BODIPY staining. Scale bar, 10 μm. Quantifications of the fluorescence intensity of lipid droplets are shown on the right. ***, P < 0.001. ns, no significance. (C-E) MARC-145 cells were treated as in A. Cellular lipids were extracted, and the amounts of TC (C), TG (D), and FFAs (E) were quantified. **, P < 0.01; ***, P < 0.001. ns, no significance.

FIG 5. EGCG inhibited the expression of key factors in lipid synthesis. (A–G) MARC-145 cells were infected with HP-PRRSV (MOI = 10) and treated with EGCG (10 μM) for 0–36 h. The mRNA levels of SREBF1 (A), FASN (B), ACACA (C), ACACB (D), SCD (E), SREBF2 (F), and HMGCR (G) were assessed by qRT-PCR analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, no significance. (H) MARC-145 cells were treated as in A. ACC, phosphorylated ACC; HMGCR, phosphorylated HMGCR. FASN, PRRSV-N, and GAPDH were assessed by immunoblotting analysis.

FIG 6. EGCG inhibited PRRSV-induced autophagy. (A) MARC-145 cells were transfected with plasmid encoding GFP-LC3 for 24 h. The cells were then mock infected or infected with HP-PRRSV (MOI = 10) and treated with EGCG (10 μM) for 36 h. GFP-LC3 puncta were detected by fluorescence microscopy. Quantification of GFP-LC3 puncta per cell is shown on the right. Scale bar, 10 μm. ***, P < 0.001. (B and C) MARC-145 cells were transfected with plasmid encoding RFP-GFP-LC3 for 24 h. The cells were then mock infected or infected with HP-PRRSV (MOI = 10) and treated with EGCG (10 μM) for 36 h. The fluorescence of GFP and RFP were detected by fluorescence microscopy (B). Scale bar, 10 μM. Quantification of autophagosomes (RFP- and GFP-positive puncta) and autolysosomes (only RFP-positive puncta) per cell are shown in C. ***, P < 0.001. (D) MARC-145 cells were infected with HP-PRRSV (MOI = 10) and treated with EGCG (10 μM) for 0–36 h. LC3-I, LC3-II, P62, PRRSV-N, and GAPDH were assessed by immunoblotting analysis. (E) Semi-quantitative densitometric analysis of LC3-II/LC3-I from D was performed with ImageJ software. ***, P < 0.001. ns, no significance. (F) Semi-quantitative densitometric analysis of…

FIG 7. Oleic acid restored PRRSV replication and assembly in response to EGCG. (A and B) MARC-145 cells were infected with HP-PRRSV (MOI = 10) and treated with EGCG (10 μM) and OA (0-150 μM) as indicated for 48 h. Viral replication was assessed with dsRNA staining (A). Quantification of the relative fluorescence intensities of dsRNA is shown in B. ***, P < 0.001; ns, no significance. (C) MARC-145 cells were treated as in A. The efficiency of viral assembly in the supernatants was determined by comparing the infectious titers (TCID50 per milliliter) with the total PRRSV genome equivalents (GE). ***, P < 0.001; ns, no significance. (D and E) MARC-145 cells were infected with PRRSV-GFP (MOI = 10) and treated with EGCG (0–10 μM) and OA (0–150 μM) as indicated for 48 h. Viral replication was analyzed by fluorescence microscopy (D). Scale bar, 100 μm. GFP-positive cells from D were analyzed by flow cytometry (E). ***, P < 0.001; ns, no significance. (F) MARC-145 cells were treated as in A. PRRSV-N and GAPDH were assessed by immunoblotting analysis. (G) Semi-quantitative densitometric analysis of PRRSV-N/GAPDH from F was performed with ImageJ software. ***, P < 0.001; ns, no significance. (H)…