The zinc-finger domain was essential for porcine reproductive and respiratory syndrome virus nonstructural protein-1α to inhibit the production of interferon-β

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused one of the most economically devastating and pandemic diseases of swine. Previous studies have documented that PRRSV nonstructural protein-1α (nsp1α) was an interferon antagonist, but the mechanism by which nsp1α inhibited the interferon (IFN)-β production was unclear. Here, by site-directed mutagenesis of the predicted zinc-coordinating residues of the zinc-finger (ZF) domain of nsp1α or by deletion of the ZF domain of nsp1α, we explored whether the ZF domain was required for nsp1α to disrupt the IFN-β production. The results showed that both mutagenesis of the predicted zinc-coordinating residues of the ZF domain and deletion of the ZF domain made nsp1α lose its interferon antagonism activity. In conclusion, our present work indicated that the ZF domain of nsp1α was necessary for nsp1α to inhibit the IFN-β induction.

FIG. 1.

The nsp1α mutant that deleted the zinc-finger (ZF) domain failed to inhibit the activities of the interferon (IFN)-β promoter (p-284 Luc) and the pIRF-3-dependent promoter (p55C1B Luc). (A) Western blots analyzed the expression of nsp1α and nsp1α 66–180 (nsp1α DZF) by anti-FLAG antibody in MARC-145 cells transfected with pcDNA3.1-FLAG (Vector), pcDNA3.1-FLAG-nsp1α (nsp1α), or pcDNA3.1-FLAG-nsp1α 66–180 (nsp1α DZF). MARC-145 cells were cotransfected with p-284 Luc (B) or p55C1B Luc (C), phRL-TK, and different expression plasmids. Twenty hours later, cells were either mock-treated (Con) or transfected with poly (I:C) for 6 h, and then the cells were harvested for the dual luciferase reporter assay. MARC-145 cells were cotransfected with p-284 Luc (D) or p55C1B Luc (E), phRL-TK, and different expression plasmids. Twenty-four hours later, the cells were harvested for dual luciferase reporter assay. Con: cells transfected with pcDNA3.1. nsp1α DZF: deletion of the ZF domain in nsp1α. Data represented means of 3 replicates, and experiments were repeated 3 times. Error bars represented the standard deviations. *P<0.05 compared with the activity of the cells with the vector.

FIG. 2.

The nsp1α mutants that mutagenesis of the predicted zinc-coordinating residues of the ZF domain failed to inhibit the activities of the IFN-β promoter (p-284 Luc) and the pIRF-3-dependent promoter (p55C1B Luc).(A) Bold italic word showed 4 residues that were proposed to coordinate zinc in EAV nsp1 and PRRSV nsp1α. (B) Western blots analyzed the expression of nsp1α, nsp1α C8A, nsp1α C10A, nsp1α C25A, and nsp1α C28A by anti-FLAG antibody in MARC-145 cells transfected with pcDNA3.1-FLAG (Vector), pcDNA3.1-FLAG- nsp1α C8A (nsp1α C8A), pcDNA3.1-FLAG-nsp1α C10A (nsp1α C10A), pcDNA3.1-FLAG-nsp1α C25A (nsp1α C25A), or pcDNA3.1-FLAG-nsp1α C28A (nsp1α C28A). MARC-145 cells were cotransfected with p-284 Luc (C) or p55C1B Luc (D), phRL-TK, and different expression plasmids. Twenty hours later, cells were either mock-treated (Con) or transfected with poly (I:C) for 6 h, and then the cells were harvested for the dual luciferase reporter assay. MARC-145 cells were cotransfected with p-284 Luc (E) or p55C1B Luc (F), phRL-TK, and different expression plasmids. Twenty hours later, the cells were harvested for the dual luciferase reporter assay. Con: cells transfected with pcDNA3.1. Data represented means of 3 replicates, and experiments were repeated 3 times. Error bars represented the standard deviations. *P<0.05 compared with the activity of the cells with the vector.

FIG. 3.

The ZF domain of nsp1α was not sufficient to inhibit the activations of the IFN-β promoter (p-284 Luc) and the pIRF-3-dependent promoter (p55C1B Luc). (A)Western blots analyzed the expression of nsp1α and nsp1α 1–65 (nsp1α ZF) by an anti-GFP antibody in MARC-145 cells transfected with pcDNA3.1-GFP (Vector), pcDNA3.1-GFP-nsp1α (GFP-nsp1α), or pcDNA3.1-GFP-nsp1α 1–65 (GFP-ZF). MARC-145 cells were cotransfected with p-284 Luc (B) or p55C1B Luc (C), phRL-TK, and different expression plasmids. Twenty hours later, cells were either mock-treated (Con) or transfected with poly (I:C) for 6h, and then the cells were harvested for the dual luciferase reporter assay. MARC-145 cells were cotransfected with p-284 Luc (D) or p55C1B Luc (E), phRL-TK, and different expression plasmids. Twenty hours later, the cells were harvested for dual luciferase reporter assay. Con: cells transfected with pcDNA3.1. GFP–nsp1α: the plasmid pcDNA3.1-GFP-nsp1α ZF that expressed nsp1α. GFP -ZF: the plasmid pcDNA3.1-GFP-nsp1α ZF that expressed ZF domain. Data represented means of 3 replicates, and experiments were repeated 3 times. Error bars represented the standard deviations. *P<0.05 compared with the activity of the cells with the vector.