Resveratrol Inhibits Pseudorabies Virus Replication by Targeting IE180 Protein

Abstract

Resveratrol is a natural polyphenolic product in red wine and peanuts and has many pharmacological activities in humans. Our previous studies showed that resveratrol has good antiviral activity against the pseudorabies virus (PRV). However, little is known about the antiviral mechanism of resveratrol against PRV. In this study, we found that resveratrol inhibited the nuclear localization of IE180 protein, which is an important step for activating early/late genes transcription. Interestingly, the results show that resveratrol inhibited the activity of IE180 protein by dual-luciferase assay. Furthermore, molecular docking analysis shows that resveratrol could bind to the Thr601, Ser603, and Pro606 of IE180 protein. Point mutation assay confirmed that resveratrol lost its inhibition activity against the mutant IE180 protein. The results demonstrate that resveratrol exerts its antiviral activity against PRV by targeting the Thr601/Ser603/Pro606 sites of IE180 protein and inhibiting the transcriptional activation activity of IE180 protein. This study provides a novel insight into the antiviral mechanism of resveratrol against herpes viruses.

Keywords: IE180 protein; antiviral; molecular docking; pseudorabies virus; resveratrol.

Figure 1

Res inhibit the expressions of pseudorabies virus (PRV) genes. Gene expression levels of PRV were quantified by RT-PCR at 1, 1.5, and 2 h in the presence or absence of Res (3.75, 7.5, and 15 μg/ml, respectively). (A) Effect of Res on the mRNA expression level of immediate early gene IE180. (B–D) Effects of Res on mRNA expression levels of early genes. (E–J) Effect of Res on mRNA expression levels of genes necessary for PRV replication. Values are presented as means ± SD (n = 3), ^*p < 0.05 vs. control group; ^**p < 0.01 vs. control group.

Figure 2

Effect of Res on IE180 protein expression. (A,B) PK-15 cells were infected with PRV (MOI = 5) for 1 h, followed by treatment with or without 15 μg/ml of Res at a different time (4, 6, and 8 h). (C,D) In the over-expression assay, the recombinant pIE180 was transfected into cells for 24 h followed by infection with PRV (MOI = 5) for 1 h. The cells were then treated with or without Res (15 μg/ml) at various times (4, 6, and 8 h). The expressions of IE180 protein were performed by western blotting and were corrected according to the levels of β-actin. Graphs show mean ± SD (n = 3).

Figure 3

The content of IE180 protein in the nucleus was decreased by Res. (A) The PK-15 cells were infected with PRV (MOI = 5) in the absence or presence of Res (15 μg/ml). Immunofluorescence was performed using anti-IE180 (green) and anti-RNA polymerase II (red). Nuclei were stained with DAPI. (B) The average fluorescence intensity ratios were calculated, and statistical analysis was performed using the Student’s t-test, ^**p < 0.01. Values are means ± SD (n = 3).

Figure 4

The effects of Res on transcriptional activation of IE180 protein by dual-luciferase assay. The 293 T cells were co-transfected with the effector plasmid pcDNA3.1(+) or pIE180, the reporter plasmid pGL3-TK, and the internal control plasmid pRL-TK. After incubation for 24 h, the Res was added to the plate to treat for 48 h, and relative luciferase activity was measured (n = 3). ^**p < 0.01 vs. control group.

Figure 5

Effects of Res on early gene expressions after transfection with pIE180. (A) Effects of Res on early gene (EPO) expressions after transfection with pIE180. (B) Effects of Res on early gene (US1) expressions after transfection with pIE180. (C) Effects of Res on early gene (UL54) expressions after transfection with pIE180.

Figure 6

The binding sites of Res with IE180 by molecular docking. (A) Alignment of the amino acid sequence of IE180 and ICP4 (5mhj.1. A). (B) The 3D structure of IE180 is conducted by homologous. (C) Ramachandran plots of the homologous mode of IE180 (Blue: the best region; Purple: the appropriate region; Red: the barely permitted region; and White: the disallowed region). (D) Profile 3D plot IE180 3D structure. (E) The 3D structure of IE180 includes an active docking site. (F) 3D docking mode between Res and IE180simulated by Discovery Studio. (G) The interaction of IE180 with Res; the green and purple colors represent H-bond and Pi-Alkyl, respectively. (H) 2D schematic interaction diagram between IE180 and Res; the green color and purple color represent H-bond and Pi-Alkyl, respectively.

Figure 7

The effects of Res on IE180 activation were detected using the dual-luciferase assay. The 293 T cells were co-transfected with the effector plasmid pcDNA3.1(+)/pIE180//pIE180^Thr601Ala/pIE180^Ser603Ala/pIE180^Pro606Ala, the reporter plasmid pGL3-TK, the internal control plasmid pRL-TK, respectively. After incubation for 24 h, the Res (15 μg/ml) was added to the plate to treat for 48 h, and relative luciferase activity was measured. ^**p < 0.01 represents a significant difference compared to the control group; ^#p < 0.01 represents a significant difference compared to the Res-pIE180 group; and ^&p < 0.01 represents a significant difference compared to the pIE180 group (n = 3, in each group).

Figure 8

Effect of Res on early gene expression after transfection with pcDNA3.1(+)/pIE180/pIE180^Thr601Ala/pIE180^Ser603Ala/pIE180^Pro606Ala. (A) Effect of Res on early gene (EPO) expression. (B) Effect of Res on early gene (US1) expression. (C) Effect of Res on early gene (UL54) expression. ^*p < 0.05 represents a significant difference compared to the pcDNA3.1(+) group (n = 3).

Figure 9

Schematic presentation of possible the Res anti-PRV molecular mechanism. Res binds to the amino acid of IE180 protein (Thr601, Ser603, and Pro606), thus affecting its transcriptional activation function, leading to the blocked downstream gene expression.