title: Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line.
abstract: Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox-)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox+)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox-)-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox-)-exo had enhanced proteolytic activity compared with HepAD38 (dox+)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox-)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox+)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox-)-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction.
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Jia
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Jieliang
authors.initials: J
authors.lastname: Chen
authors.affiliation: §Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany.
authors.forename: Dominik A
authors.initials: DA
authors.lastname: Megger
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Xiaonan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Maya
authors.initials: M
authors.lastname: Kozlowski
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Zhang
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Fang
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Qiaofang
authors.initials: Q
authors.lastname: Chu
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Min
authors.initials: M
authors.lastname: Wu
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China.
authors.forename: Yaming
authors.initials: Y
authors.lastname: Li
authors.affiliation: §Medizinisches Proteom-Center, Ruhr-Universität Bochum, 44801 Bochum, Germany 13916306983@163.com barbara.sitek@rub.de.
authors.forename: Barbara
authors.initials: B
authors.lastname: Sitek
authors.affiliation: From the ‡Shanghai Public Health Clinical Center and Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology, MOE/MOH, Shanghai Medical College, Fudan University, Shanghai 201508, China; 13916306983@163.com barbara.sitek@rub.de.
authors.forename: Zhenghong
authors.initials: Z
authors.lastname: Yuan
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D055354:Exosomes / Q000378:metabolism / Q000821:virology*; D006515:Hepatitis B virus / Q000502:physiology*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: C508600:IL6 protein, human; D015850:Interleukin-6; D014764:Viral Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease