title: Quantitative proteomic analysis of exosome protein content changes induced by hepatitis B virus in Huh-7 cells using SILAC labeling and LC-MS/MS.
abstract: Hepatitis B virus (HBV) infection could cause hepatitis, liver cirrhosis, and hepatocellular carcinoma. HBV-mediated pathogenesis is only partially understood, but X protein (HBx) reportedly possesses oncogenic potential. Exosomes are small membrane vesicles with diverse functions released by various cells including hepatocytes, and HBV harnesses cellular exosome biogenesis and export machineries for virion morphogenesis and secretion. Therefore, HBV infection might cause changes in exosome contents with functional implications for both virus and host. In this work, exosome protein content changes induced by HBV and HBx were quantitatively analyzed by SILAC/LC-MS/MS. Exosomes prepared from SILAC-labeled hepatoma cell line Huh-7 transfected with HBx, wildtype, or HBx-null HBV replicon plasmids were analyzed by LC-MS/MS. Systematic analyses of MS data and confirmatory immunoblotting showed that HBx overexpression and HBV, with or without HBx, replication in Huh-7 cells indeed caused marked and specific changes in exosome protein contents. Furthermore, specific changes in protein contents were also detected in exosomes purified from HBV-infected patients' sera compared with control sera negative for HBV markers. These results illustrate a new aspect of interactions between HBV and the host and provide the foundation for future research into roles played by exosomes in HBV infection and pathogenesis.
authors.affiliation: Key Laboratory of Medical Molecular Virology (MOE/MOH) and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University , 138 YiXueYuan Road, Shanghai 200032, People's Republic of China.
authors.forename: Xue
authors.initials: X
authors.lastname: Zhao
authors.forename: Yanxin
authors.initials: Y
authors.lastname: Wu
authors.forename: Jinlin
authors.initials: J
authors.lastname: Duan
authors.forename: Yanchun
authors.initials: Y
authors.lastname: Ma
authors.forename: Zhongliang
authors.initials: Z
authors.lastname: Shen
authors.forename: Lili
authors.initials: L
authors.lastname: Wei
authors.forename: Xiaoxian
authors.initials: X
authors.lastname: Cui
authors.forename: Junqi
authors.initials: J
authors.lastname: Zhang
authors.forename: Youhua
authors.initials: Y
authors.lastname: Xie
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology / Q000821:virology; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D055354:Exosomes / Q000378:metabolism* / Q000821:virology; D006515:Hepatitis B virus / Q000235:genetics / Q000502:physiology; D054884:Host-Pathogen Interactions; D006801:Humans; D015151:Immunoblotting; D007553:Isotope Labeling / Q000379:methods*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology / Q000821:virology; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014162:Transfection; D014779:Virus Replication / Q000235:genetics
chemical_list: D020543:Proteome
keywords: HBV; HBx; SILAC; VCP; exosome; hepatocyte; patient serum