The Study On Function Properties Of Hepatitis E Virus Open Reading Frame3 Protien

[Background]Hepatitis E is a viral hepatitis (liver inflammation) caused by hepatitis E virus (HEV) infection. This virus was first documented as infectious agent in 1983. People get more knowledge on HE with the development of scientific technology. HE is popular in net noly areas of poor sanitation but also developed areas. The disease has a poor prognosis in the context of preexisting chronic liver disease, and the mortality of hepatitis e up to 30% in pregnant women. There were chronic HE reported recently. And the reports regarding HE have an upward trend. Thus, HE become an important public health concern in worldwide. And the sutdy on HE have attracted more attention by scientist.The genome is approximately 7200 bases in length, is a polyadenylated single-strand RNA molecule that contains three discontinuous and partially overlapping open reading frames (ORFs). ORF1 encode a methyltransferase, protease, helicase and replicase which have important roles in HEV replication and transcription; ORF2 encode the capsid protein and a three-dimensional, atomic-resolution structure of the capsid protein in the context of a virus-like particle has been described; ORF3 encodes a protein of undefined function. There were four major genotypes of mammalian HEV have been reported. Genotype 1, genotype 2, genotype 3 shares the similar genome, which are different from genotype 4. As we know, the most comon genotypes are genotypel and genotype 4 in China. There is difference between genotype 1 and genotype 4 about ORF3 protein:the ORF3 protein which encoded by genotype 4 HEV is shorter than genotype 1(9 amino acids shorter at N-terminal). Till now, an in vitro culture system is not yet available. Both the incidence of hepatitis E in pathogenesis and biofunction of ORF3 coded by HEV are not clear. The report on biofunction between different genotype about ORF3 protein is not available.This research explored function of ORF3 on basis of Huh7 cell lines, and detected the role on immunity of healthy people between genotype 1 HEV and genotype 4 HEV, Which could be provided a theoretical basis for the study of hepatitis E.[Objective]Explore the influence and differences of genotype 1 and 4 ORF3 protein on Hepatocellular carcinoma cell lines respectively, and the possible mechanism. Study the influence and differences of genotype 1 and 4 HEV on immunity of health people also. To provides new scientific basis for further understand pathogenesis mechanism of hepatitis E infection.[Methods]1. Construct eukaryotic expression plasmids of gene type 1 and 4 ORF3 protein respectively. Transfect different plasmids into Huh7 cells:CCK8 detect Cell proliferation by different genotypes of ORF3 protein on Huh7 cells; Flow cytometric is used to detect cell cycle after dying with PI; cell apoptosis is explored by flow cytometry which is marked by Annexin V/PI.2. Transfect plasmids of gene type 1 and 4 ORF3 protein into Huh7 cells respectively. The expression level of gene cyclinDl and cyclinE which are related to regulate the cell cycle, are investigated by Real-time PCR, and the protein of cyclinD1,cyclinE,cyclin A,cyclin B are detected by western blot; Real-time PCR detect expression of gene Bax,Bcl-xl which are related to cell apoptosis, Western blot detect protein Bax,Bcl-x1,Caspase-9; Immunofluorescence observes influence of different types of ORF3 protein on p65 distribution and translocation, then western blot is used to confirm at protein level.3. Collect the blood of healthy people (which liver function are normal and the antibodys of hepatitis E,hepatitis B and hepatitis C are negative), separated peripheral blood mononuclear cell (PBMC) by Ficoll, and then different types of hepatitis e virus is added to PBMC. Inverted microscope is used to observe the growth state of PBMCs; INF-y and IL-4 secretion are detected by ELISA.[Results]1. Successfully construct eukaryotic expression plasmids of gene type 1 and 4 ORF3 protein; Genotype 1 ORF3 can inhibit the prolifetation activity of Huh7 cells (p< 0.05), while genotype 4 has no statistical difference; Gene type 1 ORF3 protein can prevent the cell cycle in G0/G1 phase, and the genotype 4 ORF3 protein has no obvious inhibition; Either genotype 1 ORF3 protein or genotype 4 ORF3 proteins can inhibit cell apoptosis which induced by Staurosporine.2.The results of Real-time PCR show both genotype 1 ORF3 protein and genotype 4 ORF3 protein can inhibit the expression of cyclinDl and cyclin E, western blot shows expression of proteins cyclinDl,cyclinE and CDK4,CDK6,CDK2 which are Cyclin-dependent kinase is decreased compared with the control in genotype 1, but no significantly difference in gene type 4; compared with the control, the expression of cyclinA and cyclin B which related to cell cycle S phase and G2 phase have no significantly difference in both of the genotypes of ORF3 protein.3. The result of Real-time PCR shows each genotype of ORF3 protein can inhibit the expression of gene Bax and Bcl-2. The detection of proteins relatd apoptosis shows the expression of Bax,caspase9,cytochrome C are significantly higher in the control and blank vector transfected groups which added the pro-apoptotic agents compared with the group of transfected ORF3 plasmids.4. Laser confocal shows there is no nuclear translocation about p65 after adding TNF-a in the cells which express ORF3 protein, without difference between genotype 1 and 4. The Western blot shows the expression of the nucleus p65 decreased compared with controls, and there is no significant difference genotype 1 and 4.5. As time goes by, PBMC which added different types of HEV gradually appeared wodge from a single scattered state, the number of wodge increase and the size becomes bigger especially the group which added genotype 1 HEV, while PBMC are always in a single scattered state from 4d to 9d which added nothing.6. Secretions of INF-y and IL-4 were measured using an enzyme-linked immunosorbent assay (ELISA). There are increased INF-y levels which stimulated by genotype 1 and 4 HEV. Compared to genotype 4 HEV stimuli, genotype 1 HEV group has a significantly increased interferon gamma (INF-y) values. And there is no significant difference in IL-4 secretion. The IL-4 secretion is lower than the minimum detection concentration in each group.[Conclusion]1. There are differences about influence on the Huh7 cells between the genotype 1 and 4 ORF3 protein:proliferation activity of Huh7 cells can be inhibited by genotype 1 ORF3 protein; the genotype 1 ORF3 protein is responsible for inducing cell cycle arrest in G0/ G1 phase, while the genetic type 4 ORF3 protein has no statistical difference. All types proteins can inhibit cell apoptosis induced by Staurosporine.2. Genotype 1 ORF3 protein can induces G0/G1 phase cell cycle arrest in Huh7 cells by inhibiting the expression of cyclinD1 and cyclin E; and the genotype 4 ORF3 protein has no obvious inhibition; Genotype 1 and 4 ORF3 protein inhibit cell apoptosis induced by Staurosporine through the mitochondrial pathway.3. ORF3 protein suppresses TNF-alpha-mediated p65 nuclear translocation.4. Compared with gene type 4 HEV, genotype 1 HEV can induce earlier and stronger immune response.