Pea peptide depressed the replication of porcine epidemic diarrhea virus through ROS-PERK mediated autophagy
Description
This study examined the anti-PEDV capability of PP in Vero cells. PP, at a concentration of 200 μM, was found to inhibit the replication of both PEDV hubei/2016/China strain and CV777 strain in Vero cells with concentration of 200 μM. Further investigations indicated that PP depressed the replication of virus life cycle, but not attachment and invasion into cells. In addition, PP can inhibit the generation of reactive oxygen species (ROS) induced by PEDV in Vero cells, which depressed autophagy caused by PEDV through PERK-eIF2α signal pathway. Our study expanded knowledge of bio-active PP antiviral capacity and deepened understanding of PEDV pathogenicity, which would be valuable for the development of control and prevention for PEDV. Fig 1showed that there was no clear toxic response to Vero cells when the PP concentration was 12.5–200 µM. Fig 2 revealed that the expression of N protein notably declined following PP treatment after PEDV CH/hubei/2016 strain (Fig.2 A and 2 B) and CV777 strain (Fig.2 C and 2 D) infection, indicating that PP was able to depress the replication of PEDV. Consistent with the western blot results, TCID50 assay indicated that the viral tiers of PP treated group decreased significantly (figure 2 E and figure 2 F). Compared to the control group, PP significantly decreased viral load (figure 2 G. figure 2 H). Fig 3 showed the addition of PP was performed under different treatment conditions, 18 hpi with PEDV hubei/2016 strain, cells were harvested and subject to western blot (Fig.3 A) , and TCID50 for release. The results showed that the PP had no effect on the viral adsorption (Fig.3 B and Fig.3 C), viral internalization (Fig.3 D and Fig.3 E) and viral release (Fig.3 H), but significantly inhibited the replication stage of viral protein (Fig.3F, G). Fig 4showed that the PEDV infection increased the fluorescence intensity compared to mocks, indicating that PEDV infection elevated the ROS production, while PP decreased the elevation of ROS caused by PEDV infection (Fig.4A, B), which was similar to that observed by NAC treatment. Fig 5 showed that PEDV infection significant increased KEAP 1 protein expression (Fig.5A, B), enhanced the LC 3II / LC3 B I ratio (Fig.5A, F) and phosphorylation of PERK and eIF2α (Fig.5A, C, D). However, addition of PP lowered KEAP 1 up-regulation, ratio of LC 3II / LC3 I (Fig.5A, B), phosphorylation of PERK and eIF2α induced by PEDV infection, which was consistent with NAC treatment (Fig.5A, B, C, and F). In line with NAC treatment, PP significantly depressed the replication of PEDV (Fig.5A and E).
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Quantitative Real-Time PCR Total RNA was extracted from Vero cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. After concentration determination by NanoDrop One (Thermo Fisher), 1000 ng RNA was reverse transcribed to cDNA using PrimeScript RT Master Mix (TAKARA, Japan) with the following conditions: 37 °C for 15min followed by 85 °C for 5 sec. Quantitative PCR (qPCR) was performed as previous described. Briefly, the 20 μL qPCR reaction system was made up of 1μL cDNA, 0.4 μL upstream and downstream primers respectively, 10μL Premix Ex Taq (Probe qPCR) (TAKARA, Japan), 0.4μL Probe, 7.8 μL Nuclease-free water, with the following procedure: 94 °C for 30 s, followed by 40 cycles of 94°C for 5sec, 60°C for 30 sec. Western blot Vero Cells infected by PEDV was incubated with or in the absence of PP for the indicated time were lysed with RIPA (BEYOTIME, CHINA) supplemented with protease inhibitor for 20 min on ice. The lysates were centrifuged at 12, 000 rpm at 4 ℃ for 15 min. The concentration of the proteins were measured with BCA according to the manufactures’ instructions. Samples separated by SDS-PAGE were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). After blocking in 5% nonfat milk for 1 h at room temperature, the membranes were incubated with primary antibody at 4 ℃ overnight. Then, the membranes were incubated with corresponding secondary antibody. Finally, the proteins were visualized using ECL luminescence reagents (BEYOTIME, CHINA). Protein signal intensities were normalized to GAPDH and quantified using Image J. Virus titration The samples were subjected freezing and thawing for 3 times, followed by centrifugation at 12, 000 rpm for 5 min and the supernatant was collected. Vero cells seeded to 96-well plates (Corning, USA) with a density of 104 cells/well were inoculated with 100 μL 10-fold serial dilutions (100 μL supernatant in 900 μL virus culture medium) of each virus stock. The cells were further cultured for 96 h, and fifty percent tissue culture infective dose (TCID50) was calculated according to the method of Reed and Muench. For the time-of-addition experiment, Vero cells seeded in the 24-well plates were washed with PBS and infected with PEDV at an MOI of 0.1 at 4 ℃ with PP. the invasion of PEDV into Vero cells, Vero cells were infected with PEDV at MOI of 0.1 at 4 ℃ for 1 h followed by inoculation with PP at 37 ℃ for 1 h, 18 hours after inoculation with maintenance solutions, Vero cells were subject to western blot to examine the effect of PP on the invasion of PEDV into Vero cells; for the replication of virus, Vero cells were infected with PEDV at MOI of 0.1 at 37 ℃ for 1 h followed by inoculation with maintenance solutions containing PP at 37 ℃ for 18 hours, then Vero cells were subject to western blot to examine the effect of PP on the replication of virus, and the supernatant was collected to test the effect of PP on the release of PP.