| BackgroundHepatitis E virus (HEV) was regarded as a "forgotten virus" so that there has little knowledge about HEV was written down before the1980’. In the1980’, an unexplained hepatitis was broken out among Soviet troops stationed in Afghanistan. Subsequently, the virus was screened in the volunteer’s feces, and named HEV after gene sequenced in1991. Recently, with the improvement of medical testing technology and people’s awareness of it, HEV associated public health problems have gradually become our matters. It used to think that HEV is primary available in the developing countries where have the poor sanitation, however, increased incidences of indigenous HEV infectious cases have been reported in the developed countries, such as Japan, European and American. Nowadays, there are approximately20million incident HEV infections over the world, with more than3million acute cases of hepatitis E and70,000hepatitis E-related deaths. HEV infection become available all over the world, and is the major agent of acute hepatitis because it accounted for more than50%of acute virus hepatitis. Since2012, the number of HEV infection and death is more than that of hepatitis A virus (hepatitis A virus, HAV) in China. It is thought previously that HEV infection is an acute self-limiting course, but there, in the general population, still have1-2%patients would develop into fulminate hepatitis and become death. Furthermore, HEV can result in chronic liver disease in the organ transplants, cancer chemotherapy, human immunodeficiency virus (HIV) infection and those people whose immune function is suppressed. HEV is associated with high mortality (26.9%) among pregnant women. Therefore, the situation of prevention and control about HEV is not optimistic. Although a recombinant vaccine preventing from HEV infection is available in China, the pathogenesis of HE remains unclear.The genome of HEV is a single-stranded positive-sense RNA of7.2kb, and including three open reading frames (ORFs) called ORF1, ORF2, and ORF3. To date, HEV was divided into four major genotypes, of them, both genotype Ⅰ and Ⅱ are prevalence merely in humans, and genotype Ⅲ and Ⅳ are zoonosis virus. Genotype Ⅰ and Ⅳ are the primary genotypes in China, and genotype Ⅰ of HEV infection associated with relatively high incidence of viremia and a more severe course than other genotype infections, but the mechanism is obscure. The pathogenesis research of HEV mainly focused on the analysis of HEV genome due to the limitations in cell culture systems and small animal models. The ORF1encodes the nonstructural protein of HEV, and the ORF2protein encode the viral capsid protein, but the ORF3protein is a small phosphorylated protein whose functions are not still clear.According to our previous study, the nucleus translocation of p65induced by tumor necrosis factor α (TNF-α) was deterred in HEV ORF3expressed cells. Therefore, we speculate that the viral replication environment of HEV may be optimized by ORF3protein through regulating the NF-κB signaling in cells. In the present study, we constructed the eukaryotic expression vector containing ORF3of genotype Ⅰ HEV, and examined the effection of ORF3on the NF-κB signaling in human A549lung epithelial cells (A549). We hope that these studies would explore the role of HEV ORF3in the NF-κB signaling induced by TNF-α, and classified the elucidated mechanisms of ORF3-mediated modulation of cellular processes involved in HEV infection.ObjectiveBased on our previous study, explore the role and specific mechanism of ORF3protein of genotype I HEV on the regulation of NF-κB signaling so that it can elucidate the elicit mechanism of higher viremia and more severe course when genotype I HEV infection.Methods1. The HEV ORF3fragment was PCR-amplified from the Sar-55gene, on behalf of genotype I HEV coming from Pakistan. It was cloned into the pEGFP-Nl vector by digesting with BamHI and HindⅡ to construct the eukaryotic expression vector containing HEV ORF3with green fluorescence, and then was confirmed by restriction digestion and DNA sequencing.2. A549cells were transiently transfected by the fusion protein of ORF3-GFP which was right sequence. At48h post-transfection, cells were exposed to50ng/ml of TNF-a. The level of p65in the cytoplasm or nucleus was detected with Western blot. The DNA binding activity of NF-κB was examined with electrophoretic mobility shift assay (EMS A). The level of TNF-α-induced NF-κB dependent gene expression was tested by real time PCR and enzyme linked immunosorbent assay (ELISA), respectively.3. The level of IKKP and phosphorylated IKBa in the classic NF-κB signaling were detected with Western blot. Gene chip technology (PCR Array) was applied to screen the key molecules which affected the NF-κB signaling in ORF3-GFP expressing cells, and then the above genes were verified by real time PCR and Western blot.4. Specific interference sequences targeting gene were designed and filtered out. The specific interference was transfected into A549cells, and then the luciferase activity of NF-κB was tested with luciferase reporter gene assay system.5. The symbolic protein of endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) were tested with Western blot, and observe the correlation between A20and ERS. A549cells were pretreated with inhibitor of UPR before transfecting ORF3-GFP, the luciferase activity of NF-κB induced by TNF-a was tested with luciferase reporter gene assay system.Results1. The eukaryotic expression vector containing HEV ORF3of genotype I was constructed successfully, and was confirmed by restriction digestion and DNA sequencing. The green fluorescence can be seen under the fluorescence microscope after48h from the fusion protein was transfected into A549cells. The ORF3protein can be detected with Western blot in the ORF3-GFP fusion protein expressing cells.2. We found through Western blot that p65remained in the cytoplasm when not stimulated by TNF-a, but it translocated into the nucleus in the control and GFP groups following TNF-a stimulation. However, it was only weakly translocated into the nucleus in ORF3-GFP expressing cells.3. Unlike the negative control, the GFP group and the positive control, showed a protein-DNA complex under TNF-a stimuli. Cells expressing ORF3-GFP protein lacked the complex, irrespective of TNF-a stimulation. This complex was abolished by excess cold probe, but remained unchanged with a mutant probe.4. Three NF-κB target genes, IL-1β, COX2and ICAM-1were analyzed by real time PCR, and the results showed that TNF-a-induced mRNA expression was inhibited in ORF3-GFP pretreated cells. Similar results for levels of protein expressed from these three genes were also confirmed by ELISA.5. Western blot showed that, following TNF-a stimulation, lower levels of IKKβ and phosphorylated IKBa were observed in ORF3-GFP expressing cells than those in ORF3-GFP unexposed cells. Suppressed activation of IKKp and phosphorylated IKBa were also seen in the ORF3-GFP-expressing cells compared to control.6. PCR Array exhibited that A20gene was more than10times higher in ORF3-GFP pretreated cells than control, which was confirmed by real time PCR and western blot, respectively.7. A20gene was silenced with RNAi, the inhibitory role of ORF3-GFP on TNF-a-induced NF-κB activity was abolished. It lies on the relevance between A20and GRP78in ORF3-GFP expressed cells. Given the inhibitor of UPR, the activity of TNF-a-induced NF-κB was reversed in ORF3-GFP expressed cells.Conclusions1. Open reading frame3of genotype I hepatitis E virus inhibits nuclear factor-kappa B signaling induced by tumor necrosis factor-a, and subsequently blocks the NF-κB dependent genes, which might be responsible for the higher viremia and a more severe course of genotype IHEV infection.2. Open reading frame3of genotype1hepatitis E virus can trigger the ERS and UPR which activate the NF-κB signal in the early stage, and subsequently, A20, as a downstream protein of NF-κB pathway, was enhanced and exerted a negative modulation. These results pose a possibility that ORF3maybe play a dual regulatory role for NF-κB signaling in different phase.3.There is a close link between ERS and NF-κB signal, ERS not only can activate NF-κB signal, but also regulates NF-κB signal negatively under certain circumstances. |
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