Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 4 Antagonizes Beta Interferon Expression by Targeting the NF-κB Essential Modulator

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly infectious pathogen that causes severe diseases in pigs and great economic losses to the swine industry worldwide. Type I interferons (IFNs) play a crucial role in antiviral immunity. In the present study, we demonstrated that infection with the highly pathogenic PRRSV strain JXwn06 antagonized type I IFN expression induced by poly(I·C) in both porcine alveolar macrophages (PAMs) and blood monocyte-derived macrophages (BMo). Subsequently, we showed that the inhibition of poly(I·C)-induced IFN-␤ production by PRRSV was dependent on the blocking of NF-B signaling pathways. By screening PRRSV nonstructural and structural proteins, we demonstrated that nonstructural protein 4 (nsp4), a viral 3C-like serine protease, significantly suppressed IFN-␤ expression. Moreover, we verified that nsp4 inhibited NF-B activation induced by signaling molecules, including RIG-I, VISA, TRIF, and IKK␤. nsp4 was shown to target the NF-B essential modulator (NEMO) at the E349-S350 site to mediate its cleavage. Importantly, nsp4 mutants with defective protease activity abolished its ability to cleave NEMO and inhibit IFN-␤ production. These findings might have implications for our understanding of PRRSV pathogenesis and its mechanisms for evading the host immune response. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a major agent of respiratory diseases in pigs.Like many other viruses, PRRSV has evolved a variety of strategies to evade host antiviral innate immunity for survival and propagation. In this study, we show that PRRSV nsp4 is a novel antagonist of the NF-B signaling pathway, which is responsible for regulating the expression of type I interferons and other crucial cytokines. We then investigated the underlying mechanism used by nsp4 to suppress NF-B-mediated IFN-␤ production. We found that nsp4 interfered with the NF-B signaling pathway through the cleavage of NEMO (a key regulator of NF-B signaling) at the E349-S350 site, leading to the downregulation of IFN-␤ production induced by poly(I·C). The data presented here may help us to better understand PRRSV pathogenesis.

MicroRNA-30c Modulates Type I IFN Responses To Facilitate Porcine Reproductive and Respiratory Syndrome Virus Infection by Targeting JAK1

“…The relative expression levels of miRNAs were normalized to U6. Previously described primers were used for mRNA analysis of porcine MX1, ISG15, PRRSV ORF7, and GAPDH (35,36). Other specific primers for qRT-PCR analysis are listed in Table II.…”

“…A mutant vector was constructed by mutating five seed nucleotides using a site-directed mutagenesis kit (Stratagene), following the manufacturer's instructions. NF-kB, ISRE, IRFs responding plasmids, and IFN-b promoter vector were constructed as described previously (35,36). A sequence containing three copies of the IFN-g-activated site (GAS) promoter element from the IRF1 gene, 59-CTGATTTCCCCGAAATGA-39, was inserted into pGL6 to construct the GAS promoter vector.…”

“…PRRSV infection induces poor IFN-I responses, and several PRRSV proteins, including Nsp1, Nsp2, Nsp11, N protein (29,30), and Nsp4 (36,42), were shown to antagonize IFNs. Nsp1 was identified as the most potent antagonist and is able to inhibit the production and signaling of IFN-I.…”

Nonstructural Protein 4 of Porcine Reproductive and Respiratory Syndrome Virus Modulates Cell Surface Swine Leukocyte Antigen Class I Expression by Downregulating β2-Microglobulin Transcription

“…Nsp4 is a 3C-like serine protease responsible for most nonstructural protein processing of PRRSV and plays an essential role in PRRSV infection (20). Nsp4 also impairs type I IFN expression by targeting the NF-B essential modulator (NEMO) and virus-induced signaling adaptor (VISA) (10,21), supporting Nsp4's critical role in modulating the host antiviral response. In this study, Nsp4 inhibited B2M transcription and impaired SLA-I expression on the cell surface, indicating its role in interfering with the SLA-I antigen presentation pathway and providing new insights into the functions of HP-PRRSV Nsp4.…”

“…4A and B), with Flag-Nsp4 having a greater effect than Flag-Nsp2. Furthermore, Nsp4 is a 3C-like serine protease responsible for most nonstructural protein processing and for antagonizing interferon (IFN) expression (20,21), playing an essential role in PRRSV infection, and we therefore selected Nsp4 for further studies.…”