The Mechanism Study Of Open Reading Frame 3 Of Genotype 1 Hepatitis E Virus Suppresses TLR3-induced Nuclear Factor-kappa B Signaling

Epidemics of Hepatitis E virus(HEV)are increasingly being reported.Although the disease is often sporadic and self-limited by fecal-oral transmission in developing countries.HEV infection,may also lead to acute hepatitis or,in rare cases,fulminant hepatic failure.High mortality rates of approximately 20%in pregnant women during the second and third trimesters are also observed with HEV infection,and is thus a serious threat to our society.The virus,which belongs to the genus Orthohepevirus of the family Hepeviridae,is a small,nonenveloped virus(27-34 nm in diameter)with a linear,single-stranded,plus-sense RNA genome of 7.2 kilobases consisting of three open reading frames:ORF1,ORF2 and ORF3.ORF1 encodes a nonstructural polyprotein involved in HEV replication,and ORF2 encodes the capsid protein that is responsible for virion assembly and immunogenicity.A multifunctional phosphoprotein that links intracellular transduction pathways,reduces the host inflammatory response and protects virus-infected cells is encoded by ORF3.The N-terminal half of ORF3 protein contains two hydrophobic domains,D1 and D2;the C-terminal half contains two proline-rich domains,P1 and P2.The virus can be classified into four distinct genotypes,a single known serotype.Genotypes 1 and 2 are transmitted exclusively in humans,and genotypes 3 and 4 are zoonotic.HEV genotype 1 has been implicated in large epidemics in all endemic countries.In addition,HEV genotype 1 is responsible for the highest incidence of acute hepatitis in young adults,tends to lead to higher morbidity and mortality during pregnancy,and has a substantial rate of fulminant hepatic failure.However,due to the absence of feasible animal models,the mechanism of HEV pathogenesis remains obscure.The HEV pathogenic mechanism is suggested to be substantially mediated by the immune system and,not by direct viral duplication.Recently,efficient culture systems for HEV have been established in A549 cells,which are derived from human lung cancer.Our research team has found that in A549 cells,the ORF3 protein of genotype 1 HEV inhibits the NF-?B signaling induced by TNF-?.However,the precise function of the ORF3 protein in other intrinsic signaling pathways remains unclear.Due to 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-?B signaling at the cellular level.Innate immunity has been described as the sentinel of the immune system,and this arm comprises various germline-encoded pattern recognition receptors(PRRs)that detect microbial components known as pathogen-associated molecular patterns(PAMPs).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),TLR7 and TLR8 recognize single-stranded RNA(ssRNA),and TLR9 senses unmethylated CpG DNA.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.Individual TLRs recruit myeloid differentiation primary response 88(Myd88)or TIR-domain-containing adaptor inducing interferon(IFN)-(3(TRIF,also known as TIC AM1)to initiate a specific response.MyD88 can transmit signals derived from all TLRs,except for TLR3,which utilizes the TRIF-dependent pathway.RIG-I-like receptors(RLRs),including RIG-I,Mda5 and LGP2,recognize cytoplasmic viral RNA.In response to viral genetic materials,IRF3/7-dependent production of IFN as well as NF-KB-dependent inflammatory cytokines and chemokines are induced to disrupt viral replication.The transcription factor NF-?B regulates multiple physiological functions,including the immune response,protection against apoptosis,and inflammation.The P65/P50 heterodimer is the most common activated form in TLR and RLR signaling,whereas in resting cells,the P65/P50 heterodimer is sequestered in the cytoplasm by the inhibitor of NF-?B(I?B)protein.I?B phosphorylation by the I?B kinase(IKK)complex is induced by a stimulus,leading to K48 ubiquitination of I?B and subsequent proteasomal degradation.As a result,P65 is liberated and migrates to the nucleus,where it promotes the expression of target genes.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 ultimately responsible for signal broadcasting.Ubiquitination(Ub)is a significant physiological modification of a target protein that regulates signal transduction by all receptor systems.K48-linked Ub moieties are recognized by the proteasomal system for the degradation of modified proteins,whereas K63-linked Ub promotes protein-protein interactions and signaling.In our studies,we discovered that the open reading frame 3(ORF3)protein of genotype 1 HEV downregulated the TLR3-mediated NF-?B signaling pathway.ORF3 plasmids containing green fluorescent protein(GFP)were overexpressed in A549 cells.The cells were exposed to different agonists of TLRs,which were associated with the virus nucleic acids and,cytoplasmic and nuclear fractions of P65(NF-?B subunit),and were evaluated by western blot and immunofluorescence assays.Knockdown experiments using shRNAs revealed that ORF3 protein inhibited NF-?B signaling through TLR3 signaling rather than through RIG-I signaling.This inhibition was also verified by the reduced expression of IL-8,TNF-a and IL-1?(NF-?B target genes)in ELISA and RT-PCR analyses.Meanwhile,we used a dual-luciferase reporter gene assay to identify the P2 domain of ORF3 responsible for this inhibition.Further research showed that tumor necrosis factor receptor 1-associated death domain protein(TRADD)and receptor-interacting protein kinase 1(RIP1)interacted with TLR3 in immunoprecipitation experiments.Intriguingly,the expression of TRADD was reduced and the K63-ubiquitination of RIP1 was restrained when ORF3 and Poly(I:C)co-existed.Furthermore,we found that Lys377 of RIP 1 acted as the functional ubiquitination site for the inhibitory role of ORF3.Altogether,we found in this study that the ORF3 protein of genotype 1 HEV downregulated TLR3-mediated NF-?B signaling via TRADD and RIP1.We 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.The purpose of this research provided new clues to explain how ORF3 product could foster virus replication.This research may contribute to the development of new therapeutic approaches for HEV infection.