Abstract
Hepatitis E virus (HEV) genotype 1 infection is common and can emerge as outbreaks in developing areas, thus posing a threat to public health. However, due to the absence of feasible animal models, the mechanism of HE pathogenesis remains obscure. The HEV pathogenic mechanism has been suggested to be mediated by the immune system and not by direct viral duplication. We firstly discovered that the open reading frame 3 (ORF3) protein of genotype 1 HEV downregulates TLR3-mediated NF-κB signaling in Human A549 Lung Epithelial Cells (A549 cells) which were exposed to different TLR agonists associated with viral nucleic acids. Additionally, we identified the P2 domain of ORF3 as being responsible for this inhibition. Intriguingly, tumor necrosis factor receptor 1-associated death domain protein (TRADD) expression and receptor-interacting protein kinase 1 (RIP1) K63-ubiquitination were reduced in the presence of both ORF3 and Poly(I:C). Furthermore, we found that Lys377 of RIP1 acts as the functional ubiquitination site for ORF3-associated inhibition. Overall, we found that ORF3 protein downregulates TLR3-mediated NF-κB signaling via TRADD and RIP1. Our findings provide a new perspective on the cellular response in HEV infection and expand our understanding of the molecular mechanisms of HEV pathogenesis in innate immunity.
Highlights
The absence of a feasible in vivo model, the purpose of our research was to verify the role of genotype 1 Hepatitis E virus (HEV) open reading frame 3 (ORF3) protein in TLR-induced NF-κB signaling at the cellular level
To observe the role of the ORF3 protein in TLR signaling, we chose to examine TLR3, TLR7, TLR8 and TLR9, which are related to the detection of single-stranded RNA (ssRNA) viruses
The dual-luciferase reporter gene system showed that Poly(I:C) could induce NF-κB activity in the GFP group and that ORF3 alone had no effect on NF-κB activity
Summary
The absence of a feasible in vivo model, the purpose of our research was to verify the role of genotype 1 HEV ORF3 protein in TLR-induced NF-κB signaling at the cellular level. TLRs were the first PRRs to be characterized, and nucleic acids derived from viruses act as agonists for multiple TLRs. For example, TLR3 detects double-stranded RNA (dsRNA)[18], TLR7 and TLR8 recognize single-stranded RNA (ssRNA), and TLR9 senses unmethylated CpG DNA19. TLR3 detects double-stranded RNA (dsRNA)[18], TLR7 and TLR8 recognize single-stranded RNA (ssRNA), and TLR9 senses unmethylated CpG DNA19 In unstimulated cells, these TLRs are located in the endoplasmic reticulum (ER); upon activation, they are translocated via the Golgi apparatus to endosomes, where they are processed by proteases to encounter the internalized nucleic acid ligands[20,21]. P65 is liberated and migrates to the nucleus, where it promotes the expression of target genes[27] During this event, TRADD and RIP1 play a crucial role in the inhibition of ORF3, whereby TRADD acts as the connector and RIP1 as an executor that is responsible for signal broadcasting. The objective of this research was to elucidate the function of the ORF3 product and to expand our understanding of the cellular processes of HEV pathogenesis
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