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

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.

HEV ORF3 downregulatesCD14 and CD64 to impair macrophages phagocytosis through inhibiting JAK/STAT pathway

“…The ORF3 of HEV encodes a phosphoprotein that can play a critical role in regulating a series of host cell processes to create an immunosuppressive environment suitable for virus survival and persistent infection . Our previous study showed that the HEV ORF3 could obviously inhibit the secretion of inflammation factors of macrophages by inhibiting the activation of nuclear factor κ‐light‐chain‐enhancer of activated B‐cells pathway .…”

Distinct Entry Mechanisms for Nonenveloped and Quasi-Enveloped Hepatitis E Viruses

“…ORF2 encodes a 72-kDa protein that forms the capsid. ORF3 encodes a small multifunctional protein (12)(13)(14)(15). Although originally recognized as a nonenveloped virus, recent studies show that HEV circulating in blood is cloaked in host membranes (16).…”

Clinical features and determinants of chronicity in hepatitis E virus infection

“…Certain TNF‐α and IFN‐γ gene promoter polymorphisms, which seem to be associated with higher cytokine production, seem to be associated with higher susceptibility for HEV infections (‐308‐AA in promoter region) or severe symptomatic disease (TNF‐1031CC and IFN‐874TT) . HEV ORF‐3 inhibits TNFα‐induced nuclear factor‐kappa B signalling . Therapy with TNF‐α inhibitors for rheumatologic conditions has been associated with HEV infection .…”

“…36 HEV ORF-3 inhibits TNFα-induced nuclear factorkappa B signalling. 37 Therapy with TNFα inhibitors for rheumatologic conditions has been associated with HEV infection. 9 Though most of these have been acute infections, chronic HEV has been described, despite anti-HEV T-cell responses being preserved, with use of these agents.…”