title: Evaluation of hepatitis B virus replication and proteomic analysis of HepG2.2.15 cell line after cyclosporine A treatment.
abstract: OBJECTIVE
  The effect of cyclosporine A (CsA) on hepatitis B virus (HBV) replication was investigated, and proteomics expression differentiation after CsA treatment was studied in order to provide clues to explore the effect of CsA on HBV replication.
  
  
  METHODS
  Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxicity of CsA. The HBV replication level in the HBV genomic DNA transfected HepG2.2.15 cell line was determined by an ELISA analysis of hepatitis B surface antigens (HBsAg) and Hepatitis B e antigens (HBeAg) in culture supernatant, while the intracellular HBV DNA replication level was analyzed by slot blot hybridization. Two-dimensional electrophoresis was used to investigate the alteration of protein expression in HepG2.2.15 after CsA treatment in vitro. The differentially-expressed proteins were identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with an online database search.
  
  
  RESULTS
  CsA was able to inhibit the expression of HBsAg, HBeAg, and HBV DNA replication in vitro in a dose-dependent manner. A proteomics analysis indicated that the expression of 17 proteins changed significantly in the CsA treatment group compared to the control group. Eleven of the 17 proteins were identified, including the overexpression of eukaryotic translation initiation factors (eIF) 3k, otubain 1, 14.3.3 protein, eIF2-1 alpha, eIF5A, and the tyrosine 3/tryptophan 5-mono-oxygenase activation protein in CsA-treated HepG2.2.15 cells. The downregulation of the ferritin light subunit, erythrocyte cytosolic protein of 51 kDa (ECP-51), stathmin 1/oncoprotein, adenine phosphoribosyl-transferase, and the position of a tumor protein, translationally controlled 1, was shifted, suggesting it had undergone posttranslational modifications.
  
  
  CONCLUSIONS
  Our study identified the inhibitory effect of CsA on HBV replication, and found that a group of proteins may be responsible for this inhibitory effect.
authors.affiliation: Department of Hepatobiliary Pancreatic Surgery, Key Laboratory of Multiple Organ Transplantation, Ministry of Public Health, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China.
authors.forename: Hai-Yang
authors.initials: HY
authors.lastname: Xie
authors.forename: Wei-Liang
authors.initials: WL
authors.lastname: Xia
authors.forename: Chun-Chao
authors.initials: CC
authors.lastname: Zhang
authors.forename: Li-Ming
authors.initials: LM
authors.lastname: Wu
authors.forename: Hao-Feng
authors.initials: HF
authors.lastname: Ji
authors.forename: Yu
authors.initials: Y
authors.lastname: Cheng
authors.forename: Shu-Sen
authors.initials: SS
authors.lastname: Zheng
mesh_terms: D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D020543:Proteome / Q000032:analysis*; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D004791:Enzyme Inhibitors; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D020543:Proteome; D016572:Cyclosporine