The ORF3 Protein Of Hepatitis E Virus Activates The Phosphatidylinositol-3-kinase Signal Transduction Pathway

ObjectiveHepatitis E is an acute infectious disease caused by hepatitis E virus (HEV). Hepatitis E is endemic in many developing countries of Asia, Africa, mid-America. Many outbreaks have been reported in Mexico, India, Pakistan, Nepal. In endemic areas, HEV infection has been showed to be a significant cause of fulminant liver,an acute and rapidly progressing form of liver disease with high fatality rate. The case-fatality rate of hepatitis E has been reported as high as 15%～25% in pregnant women. An outbreak occurred in Xinjiang Autonomous Region, China, in 1986 and 1988. It consisted of 110000 cases and caused 1000 people's death nearly. There are more and more reported cases of hepatitis E in the nearest years in China. Hepatitis E has become an important disease in many countries around the world.The pathogenesis and molecular biology of HEV are poorly understood due to the lack of a reliable in vitro culture system or small animal models of infection. The cause of high fatality rate in pregnant women with hepatitis E is also not understood. There is no good cure in treating hepatitis E as to now. It is very important to explore the pathogenesis furtherly for treating this disease with new cure.The HEV genome is a 7.2～7.5-kilobase positive-sense RNA that contains three open reading frames (ORFs) designed ORF1, ORF2, and ORF3. The ORF3 of HEV encodes a protein of ～13.5kD, called pORF3, for which no function has been assigned. When express in animal cell, pORF3 is phosphorylated at a single serine residue in its 123-amino acid primary sequence. In vitro phosphorylation experiments suggested thatpORF3 may be a substrate of the mitogen-activated protein kinase (MAPK), and subcellular fractionation revealed it association with cytoskeleton. These observations suggest a possible role for pORF3 in the signal transduction pathway. Another clue that pORF3 may have a potential role in cell signaling is the presence of praline-rich (PXXP) sequences, which are conserved among different isolates of HEV around the world. Such PXXP motifs are part of polyproline helices found in many proteins involved in signal transduction and bind the Src homology 3 (SH3) domains found in many signal transducting molecules.Abnormal signal transduction play important roles in a variety of diseases including infectious diseases. A number of viral proteins have been known to bind host cell proteins to either activate or in activate signal transducting molecules for the benefit of virus replication, infection persistence, or evasion from host immune responses. The purpose of this study is to explore the possible role of pORF3 in the modulation of the phosphatidylinositol-3-kinase signal transduction pathway.Methods1. HEV RNA was extracted from the bile of macaque experimental infected with HEV by using Trizol kit according to the manufacturer's guidelines; HEV particles were found in the bile of macaque through immune electron microscopy;2. HEV cDNA was synthesized through reverse-transcription reaction with HEV RNA as template;3. HEV ORF3 cDNA was synthesized through polymerase chain reaction with HEV cDNA as template;4. HEV ORF3 cDNA was extracted from the gel using QIAquick Gel Extraction kit according to the manufacturer's guidelines after agar gel electrophoresis;5. Plasmid pcDNA3 and HEV ORF3 cDNA were cleaved with restriction endonuclease EcoR â… /Xho â… ;6. Plasmid pcDNA3 and HEV ORF3 cDNA were ligated to construct recombinant(pcHEV3) with T4 DNA ligase after which cleaved with EcoR â… /Xho â… ;7. Competent Escherichia coli (Amp⁺) were transformed with recombinants(pcHEV3) into recombinant bacteria;8. Recombinants (pcHEV3) were cleaved with restriction endonuclease EcoR â… /Xho â…  to carry on agar gel electrophoresis after extracted from the recombinant bacteria obtaining recombinants (pcHEV3);9. HEV ORF3 cDNA bases were sequenced in recombinants (pcHEV3) by use of automatic DNA sequencing instrument;10. HepG2 cells were cultured in DMEM containing 10% fetal bovine serum and were transiently transfected with recombinants (pcHEV3) by using Lipofect AMINE according to the manufacturer's guidelines;11. At about 48 h posttransfection, HepG2 cells were fixed in acetone. Then cells were stained with anti-pORF3 polyclonal antibodies and anti-mouse antibodies conjugated with FITC. Stained cells were observed with an epifluorescence microscope;12. At about 48 h posttransfection, HepG2 cells were dissolved in 1%NP-40 buffer containing phosphatase inhibitors (50 mM NaF and 0.2 mM Na₃VO₄) and protease inhibitors (0.1 mM PMSF, 1 μM pepstatin, 0.5 mg/ml leupeptin and 0.3 μM aprotinin), supernatant of lysates were subjected to western blot for phosphorylated Akt;13. At about 48 h posttransfection, HepG2 cells were dissolved in hyposmolar lysis buffer containing protease inhibitors (0.1 mM PMSF, 1 μM pepstatin, 0.5 mg/ml leupeptin and 0.3 μM aprotinin), the nuclear extracts were subjected to electrophoretic mobility shift assay to detect the activation and nuclear translocation of nuclear factor-κB.Results1. The results show that HEV ORF3 cDNA is cloned from the bile of macaque experimental infected with HEV by use of reverse transcription-polymerase chainreaction (RT-PCR);2. The experiments indicate that recombined expression vector (pcHEV3) is constructed successfully;3. The data confirm that HEV ORF3 protein is exepressed in the cytoplasm of HepG2 cells after which transfected with recombined plasmid;4. The results demonstrate that phosphorylated Akt is increased in HepG2 cells after which transfected with recombined plasmid; Furthermore, phosphorylated Akt is depended on the expression of HEV ORF3 protein;5. It is concluded that nuclear translocation NF-κB is translocated into the nucleus of HepG2 cells which depend on the aitivation of phosphatidylinositol-3-kinase signal transduction pathway.Conclusion1. The experiments indicate that the phosphatidylinositol-3-kinase is activated in HepG2 cells after which transfected with recombined plasmid and exepressed of hepatitis E virus open reading frame 3 protein;2. It is concluded that the hepatitis E virus open reading frame 3 protein activates the phosphatidylinositol 3-kinase-Akt signal transduction pathway;3. The results show that the hepatitis E virus open reading frame 3 protein regulates the nuclear translocation of NF-κB through the activation of phosphatidylinositol-3-kinase signal transduction pathway;4. It is concluded that phosphatidylinositol-3-kinase signal transduction pathway is blocked by use of LY294002;5. It is suggested that blockage of the phosphatidylinositol-3-kinase signal transduction pathway could be new therapeutic target for the treatment of hepatitis E.