Investigation On Interactions Between Protein Encoded By 2447nt-489nt Spliced Variant Of Hepatitis B Virus Genome And Hepatocellular Proteins

Spliced variants of hepatitis B virus genomes are a group of subgenomic length DNA generated from 3.5 kb pregenomic RNA by splicing and reverse transcription. More than 80% of these spliced variants are 2.2 kb singly spliced variants of hepatitis B virus genome (spliced at the nucleotides position 2447 nt-489 nt, also called 2447 nt-489 nt HBV spliced variant), which could encode Hepatitis B spliced protein (HBSP) and closely related with persistent infection and pathogenicity of HBV. We had screened for hepatocellular proteins interacted with HBSP by yeast two-hybrid system and identified fibrinogen gamma chain (FGG) and microsomal epoxide hydrolase (mEH, encoded by EPHX1). The aim of this study was to further verify the interactions between HBSP and FGG or mEH, and elucidate the influences of HBSP on hemostatic and liver biotransformation.The first part of this study is to confirm the interactions of HBSP with FGG or mEH. To address these issues, the plasmids of pGEX-HBSP (used for GST pull-down), pDsRed-HBSP (used for confocal microscopy), pBIND-HBSP, pBIND-HBSP1-47 and pBIND-HBSP64-111 (used for mammalian two-hybrid assay and co-immunoprecipitation) were constructed, also, the full length of FGG and EPHX1 cDNA were amplified by RT-PCR and the recombinant plasmids used for in vitro translation (pCMVTNT-FGG and pCMVTNT-EPHX1) and confocal microscopy (pAcGFP-FGG and pAcGFP-EPHX1) were constructed. It was demonstrated that HBSP could interact with FGG and mEH in vitro and in vivo, and these interactions are mediated by the N terminal 47 amino acid residues of HBSP.The second part of this study is to obtain a large amount of HBSP fusion proteins with high purity. Therefore, the full length, the N terminal region (encode N terminal 47 amino acid residues) and C terminal region (encode C terminal 64 amino acid residues) of HBSP gene were respectively cloned into the prokaryotic expression vector to construct plasmids of pET-43-HBSP-StrepII, pET-43-HBSP1-47-StrepII, pET-43-HBSP48-111-StrepII and pET-43-StrepII, respectively. The results showed that the fusion proteins including Nus-HBSP-StrepII, Nus-HBSP1-47-StrepII, Nus-HBSP48-111-StrepII and Nus-StrepII could be expressed with high level and solubility, and the fusion proteins with high purity could be obtained after Strep-Tactin affinity chromatography.The third part of this study is aimed to elucidate the influences of HBSP on the functions of FGG and mEH. It was revealed that HBSP could inhibit fibrin polymerization, interfere the adhesion of platelets to fibrinogen and ADP-stimulated platelet aggregation, thus inhibit haemostatic. It was also confirmed that HBSP could greatly enhance the activity of mEH, thus accelerate the metabolic of chemical carcinogen of Benzo[a]pyrene to produce more ultimate carcinnogen of Benzo(a)pyrene-7r,8s-dihydrodiol-9s,10r-epoxide(±)(BPDE). Taken together, it suggested HBSP may participate in the haemostatic abnormality in patients with HBV related liver diseases, and increase the carcinogenic effect of PAHs (Polyeyclie Aromatie Hydroearbons).