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PLoS One. 2014 Jun 24;9(6):e100787. doi: 10.1371/journal.pone.0100787. eCollection 2014.

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.

Author information

1
Department of Infectious Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
2
Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
3
National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Public Health, Xiamen University, Xiamen, China.

Abstract

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.

PMID:
24959724
PMCID:
PMC4069097
DOI:
10.1371/journal.pone.0100787
[Indexed for MEDLINE]
Free PMC Article

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