The Roles Of Open Reading Frame 3 Protein In HEV-related Immune Evasion And Its Mechanisms

Hepatitis E virus(HEV)infection is a worldwide public health problem,which caused around 20 million new infections worldwide each year.Despite the acute self-limitation course of hepatitis E,more and more reports indicated that HEV might leads to fulminant hepatic failure or chronic hepatitis E in some special patients such as organ transplantation,HIV and pregnant women.Since the high fatality rate in cirrhosis patients and pregnant women is not well described,further studies are urgently needed.Macrophage is the most important component of the innate immune system.Macrophage can not only swallow and secrete a variety of inflammatory factors to destroy the invading pathogens,but also play an important role in antigen presenting to link the innate immune and specific immune responses.At the same time,virus infection can also activate the innate anti-viral immune responses by activating the pattern recognition receptors.But mostly viruses have evolved a variety of strategies to evade the host's immunity.The present researches confirmed that the open reading frame 3 of HEV(HEV ORF3)can interact with the host cell proteins to create a good environment for HEV replication and pathological progress.Therefore,we hypothesized that HEV ORF3 may inhibit the immune responses and the phagocytosis of macrophages,and further induces chronic hepatitis E or even liver fibrosis and cirrhosis.In addition,HEV ORF3 may also downregulate the pattern recognition receptors to inhibit the antiviral immune responses in host cells and maintain the continuous replication status of HEV by escaping the immune clearance.Objective:To construct HEV ORF3-overexpressing vectors and establishes the appropriate conditions in different cell models.To clarify the regulation of HEV ORF3 on macrophage polarization and the release of inflammatory factors and chemokines.Study the effects of HEV ORF3 on phagocytosis and phagocytic receptors in macrophages.And explore the regulation effects and mechanisms of HEV ORF3 on pattern recognition receptors and generation of type I endogenous interferon.Methods:Various overexpression vectors of HEV ORF3 protein including lentivirus,adenovirus and plasmid vector were constructed.THP1 cells were differentiated by PMA.The appropriate host cell model was selected according to the expression level of TLR7 in L02,HepG2 and Huh7 cell lines.LPS was added to induce the inflammation in PMA-THP1 macrophages infected by lentivirus or adenovirus.The effects of HEV ORF3 on macrophages was shown by using ELISA and real-time quantitative PCR,Western blot methods.Furthermore,the activity of NF-kappa B pathway was tested to figure out the reason for regulation of inflammatory factors and chemokines.Flow cytometry and phagocytosis test were constructed in order to clarify the inhibition of phagocytosis by HEV ORF3.Further results of RT-qPCR,Western blot was used to figure out the related receptors in this progress.And the mechanism of this phenomenon was tested by combination with agonists and inhibitors.Levels of type I endogenous interferon in PMA-THP1 macrophage and L02 cells was studied by over-expressing HEV ORF3.Further tests of RT-qPCR,Western blot were managed to find the responding pattern recognition receptors.And its mechanism was then explored by combining the specific signaling pathway regulators.Results:The multiply vectors of genotype 1 HEV ORF3 including lentivirus,adenovirus and plasmid vector were successfully constructed.THP1 could be differentiated by treating with 1Ong/mL PMA for 24h,and then cultured for another 24h in complete medium.L02 cell line was selected the benefit cell model because of its higher expression of TLR7 protein compared with HepG2 and Huh7 cell lines.Results of ELISA and RT-qPCR indicated that ORF3 could significantly impair the generation of inflammatory factors and chemokines induced by lug/mL LPS in PMA-THP1 cells.And the downregulation of TLR4 and inhibition of NF-kappa B pathway play important role in its inhibition.Results of flow cytometry and phagocytosis test found that HEV ORF3 can obviously inhibit the phagocytosis of macrophage.RT-qPCR and western blot showed that expression of CD 14 and CD64 were significantly reduced,which could be reversed by pretreating with ruxolinitib,a specific inhibitors of JAK/STAT signaling pathway.At last,our results of ELISA and RT-qPCR indicated that ORF3 could significantly inhibit the production of type I interferon both in PMA-THP1 and L02 cells.Further results showed that the expression of TLR3 and TLR7 were reduced in ORF3-expressing cells.Interesting,we found that the inhibition of TLR7 by HEV ORF3 might function by inhibiting the expression of multiply factors including p-P65,p-STAT1 and p-JNK.Conclusion:Our results indicated that HEV ORF3 could significantly reduce the expression of inflammatory and Chemokines factors in PMA-THP1 macrophages induced by LPS,which might result by down-regulating TLR4 and inhibiting the activity of NF-kappa B signaling pathway.We also found that HEV ORF3 could inhibit the expression of phagocytic receptors including CD14 and CD64 by inhibiting JAK1/STAT1 signaling pathway,furthermore inhibit the phagocytosis.What's more,HEV ORF3 could aslo inhibit the generation of type I endogenous interferon by down-regulating TLR3 and TLR7.And the downregulation of TLR7 was reduced by inhibiting multiple signaling pathways including NF-kappa B,JAK/STAT and JNK/MAPK.So we suppose that HEV ORF3 plays crucial role in immune evasion of HEV by multiple strategies,and our results provide a solid theoretical supportment and supplementation for the mechanism of chronic hepatitis E.