Research On The Nf-?b Pathway Regulation By ORF3 Of Genotype 3 Hepatitis E Virus

Hepatitis E virus(HEV)infection is one of the most common causes of acute hepatitis or sporadic acute hepatitis in many parts of the world.At least 20 million HEV infections occur annually,which are mostly self-limiting and have different clinical manifestations,such as asymptomatic,acute liver failure and rare chronicity.In some cases,HEV infections can cause up to 30% mortality in pregnant women and may result in chronic hepatitis in immunocompromised individuals,such as those receiving organ transplants or chemotherapy,and individuals infected with HIV.Currently,at least four genotypes exist among HEV strains,of which HEV genotypes 1 and 2 are obligate human pathogens and HEV genotypes 3 and 4 are mostly zoonotic.HEV is a 7.2-kb single-stranded,positive-sense RNA genome containing three open reading frames(ORFs).The ORF1 protein comprises the functional domains of virions;ORF2 is the major structural protein of virions and encodes the viral capsid protein;andORF3 protein,a small molecule phosphoprotein,is involved in viral particle secretion.A recent study found that chronic HEV infections were almost exclusively caused by the zoonotic genotype 3 HEV strains.Innate immunity is the first line of defense against viruses and is mainly dependent on host pattern recognition receptors(PRRs).The PRRs for RNA viruses include the retinoic-acid-inducible gene I(RIG-I)-like receptor(RLR)and the TLR.Both RLR and TLR3 can recognize viral genomic double-stranded RNA.The host cells can sense the internalized virus through the TLRs on the endosomal membrane,whereas the RLRs mainly sense the virus that enters the cytoplasm.TLR signal transduction pathways are divided into the human myeloid differentiation factor 88(MyD88)-dependent pathway and the MyD88-independent pathway.The MyD88 pathway mediates TLR1,TLR2,TLR5,TLR6,TLR7,TLR8,and TLR9 signals,while the toll-interleukin 1 receptor homology-domain-containing adapter-inducing interferon-?(TRIF)pathway mediates TLR3 signaling.The RLR family consists of RIG-I and melanoma differentiation-associated gene 5(MDA5).Activation of TLR and signaling leads to the activation of NF-? B and interferon(IFN)pathways,which can induce the expression of numerous cytokines and inflammatory factors,and initiate innate immune responses to resist virus infection.Activation of NF-?B is a hallmark of many viral infections.After activation of NF-?B,it activates a variety of cytokines and chemokines(such as TNF-?,IL-6).These activatedcytokines and chemokines can activate the NF-?B pathway again,resulting in increased production and release,leading to further amplification of the initial inflammatory signal.During viral infection,NF-?B activation is considered to be a protective response of the host to the pathogen.Innate immune activation regulates important components such as cytokines,chemokines,adhesion molecules,and enzymes that produce inflammatory mediators and inhibitors of apoptosis,and transcription of genes encoding these molecules is associated with NF-?B activation.At the same time,studies have reported that activation of NF-?B may be a mechanism that inhibits apoptosis and prolongs host cell survival in order to gain replication and increase progeny virus production.Our previous study found that genotype 1 HEV ORF3 inhibited both TNF? and TLR3-mediated NF-?B signaling pathways,but there was no report on the regulation of NF-?B signaling pathway by genotype 3 HEV ORF3.In this study,we first investigated the effects of ORF3 of genotype 3 HEV on NF-?B signaling.We have shown that the ORF3 protein activates the NF-?B pathway induced by Poly(I:C).The P2 domain of ORF3 was found to be responsible for this activation.HEV ORF3 activated NF-?B P65 via the TRIF-dependent TLR3 pathway.The expression of the ORF3 protein led to an increased level of TLR3 by regulating its transcription level and extending its half-life.Similarly,The expression of the ORF3 protein led to an increased TRIF level by regulating its transcription level and extending its half-life.These results indicate that the ORF3 protein could activate the NF-?B pathway via TLR3-TRIF pathway.