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Open Reading Frame 3 of Genotype 1 Hepatitis E Virus Inhibits Nuclear Factor-κappa B Signaling Induced by Tumor Necrosis Factor-α in Human A549 Lung Epithelial Cells

Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis, and represents a major cause of severe public health problems in developing countries. The pathogenesis of HEV is not well characterized, however, primarily due to the lack of well-defined cell and animal models. Here...

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Autores principales: Xu, Jian, Wu, Fan, Tian, Deying, Wang, Jingjing, Zheng, Zizheng, Xia, Ningshao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069097/
https://www.ncbi.nlm.nih.gov/pubmed/24959724
http://dx.doi.org/10.1371/journal.pone.0100787
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author Xu, Jian
Wu, Fan
Tian, Deying
Wang, Jingjing
Zheng, Zizheng
Xia, Ningshao
author_facet Xu, Jian
Wu, Fan
Tian, Deying
Wang, Jingjing
Zheng, Zizheng
Xia, Ningshao
author_sort Xu, Jian
collection PubMed
description Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis, and represents a major cause of severe public health problems in developing countries. The pathogenesis of HEV is not well characterized, however, primarily due to the lack of well-defined cell and animal models. Here, we investigated the effects of genotype 1 HEV open reading frame 3 (ORF3) on TNF-α-induced nucleus factor-κappa B (NF-κB) signaling. Human lung epithelial cells (A549) were transiently transfected with ORF3 containing plasmids. These cells were then stimulated with TNF-α and the nucleus translocation of the p65 NF-κB subunit was assessed using western blot and laser confocal microscopy. DNA-binding activity of p65 was also examined using electrophoretic mobility shift assay (EMSA), and the suppression of NF-κB target genes were detected using real-time RT-PCR and ELISA. These results enabled us to identify the decreased phosphorylation levels of IKBα. We focused on the gene of negative regulation of NF-κB, represented by TNF-α-induced protein 3 (TNFAIP3, also known as A20). Reducing the levels of A20 with siRNAs significantly enhances luciferase activation of NF-κB. Furthermore, HEV ORF3 regulated A20 primarily via activating transcription factor 6 (ATF6), involved in unfolded protein response (UPR), resulting in the degradation or inactivation of the receptor interacting protein 1 (RIP1), a major upstream activator of IKB kinase compounds (IKKs). Consequently, the phosphorylation of IKBα and the nucleus translocation of p65 are blocked, which contributes to diminished NF-κB DNA-binding activation and NF-κB-dependent gene expression. The findings suggest that genotype 1 HEV, through ORF3, may transiently activate NF-κB through UPR in early stage, and subsequently inhibit TNF-α-induced NF-κB signaling in late phase so as to create a favorable virus replication environment.
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spelling pubmed-40690972014-06-27 Open Reading Frame 3 of Genotype 1 Hepatitis E Virus Inhibits Nuclear Factor-κappa B Signaling Induced by Tumor Necrosis Factor-α in Human A549 Lung Epithelial Cells Xu, Jian Wu, Fan Tian, Deying Wang, Jingjing Zheng, Zizheng Xia, Ningshao PLoS One Research Article Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis, and represents a major cause of severe public health problems in developing countries. The pathogenesis of HEV is not well characterized, however, primarily due to the lack of well-defined cell and animal models. Here, we investigated the effects of genotype 1 HEV open reading frame 3 (ORF3) on TNF-α-induced nucleus factor-κappa B (NF-κB) signaling. Human lung epithelial cells (A549) were transiently transfected with ORF3 containing plasmids. These cells were then stimulated with TNF-α and the nucleus translocation of the p65 NF-κB subunit was assessed using western blot and laser confocal microscopy. DNA-binding activity of p65 was also examined using electrophoretic mobility shift assay (EMSA), and the suppression of NF-κB target genes were detected using real-time RT-PCR and ELISA. These results enabled us to identify the decreased phosphorylation levels of IKBα. We focused on the gene of negative regulation of NF-κB, represented by TNF-α-induced protein 3 (TNFAIP3, also known as A20). Reducing the levels of A20 with siRNAs significantly enhances luciferase activation of NF-κB. Furthermore, HEV ORF3 regulated A20 primarily via activating transcription factor 6 (ATF6), involved in unfolded protein response (UPR), resulting in the degradation or inactivation of the receptor interacting protein 1 (RIP1), a major upstream activator of IKB kinase compounds (IKKs). Consequently, the phosphorylation of IKBα and the nucleus translocation of p65 are blocked, which contributes to diminished NF-κB DNA-binding activation and NF-κB-dependent gene expression. The findings suggest that genotype 1 HEV, through ORF3, may transiently activate NF-κB through UPR in early stage, and subsequently inhibit TNF-α-induced NF-κB signaling in late phase so as to create a favorable virus replication environment. Public Library of Science 2014-06-24 /pmc/articles/PMC4069097/ /pubmed/24959724 http://dx.doi.org/10.1371/journal.pone.0100787 Text en © 2014 Xu et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Xu, Jian
Wu, Fan
Tian, Deying
Wang, Jingjing
Zheng, Zizheng
Xia, Ningshao
Open Reading Frame 3 of Genotype 1 Hepatitis E Virus Inhibits Nuclear Factor-κappa B Signaling Induced by Tumor Necrosis Factor-α in Human A549 Lung Epithelial Cells
title Open Reading Frame 3 of Genotype 1 Hepatitis E Virus Inhibits Nuclear Factor-κappa B Signaling Induced by Tumor Necrosis Factor-α in Human A549 Lung Epithelial Cells
title_full Open Reading Frame 3 of Genotype 1 Hepatitis E Virus Inhibits Nuclear Factor-κappa B Signaling Induced by Tumor Necrosis Factor-α in Human A549 Lung Epithelial Cells
title_fullStr Open Reading Frame 3 of Genotype 1 Hepatitis E Virus Inhibits Nuclear Factor-κappa B Signaling Induced by Tumor Necrosis Factor-α in Human A549 Lung Epithelial Cells
title_full_unstemmed Open Reading Frame 3 of Genotype 1 Hepatitis E Virus Inhibits Nuclear Factor-κappa B Signaling Induced by Tumor Necrosis Factor-α in Human A549 Lung Epithelial Cells
title_short Open Reading Frame 3 of Genotype 1 Hepatitis E Virus Inhibits Nuclear Factor-κappa B Signaling Induced by Tumor Necrosis Factor-α in Human A549 Lung Epithelial Cells
title_sort open reading frame 3 of genotype 1 hepatitis e virus inhibits nuclear factor-κappa b signaling induced by tumor necrosis factor-α in human a549 lung epithelial cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069097/
https://www.ncbi.nlm.nih.gov/pubmed/24959724
http://dx.doi.org/10.1371/journal.pone.0100787
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