title: Two-dimensional blue native/SDS-PAGE analysis reveals heat shock protein chaperone machinery involved in hepatitis B virus production in HepG2.2.15 cells.
abstract: Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60, HSP70, and HSP90 proteins physically interacted in HepG2.2.15 but not HepG2 cells. We further demonstrated that down-regulation of HSP70 or HSP90 by small interfering RNA significantly inhibited HBV viral production but did not influence cellular proliferation or apoptosis. Consistent with these results, a significant reduction in HepG2.2.15 HBV secretion was observed when the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin was used to treat HepG2.2.15 cells. Collectively these results suggest that the interaction of HSP90 with HSP70/HSP60 contributes to the HBV life cycle by forming a multichaperone machine that may constitute therapeutic targets for HBV-associated diseases.
authors.affiliation: State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Liu
authors.forename: Lu
authors.initials: L
authors.lastname: Qian
authors.forename: Jinglan
authors.initials: J
authors.lastname: Wang
authors.forename: Wenrui
authors.initials: W
authors.lastname: Li
authors.forename: Xinyu
authors.initials: X
authors.lastname: Deng
authors.forename: Xilin
authors.initials: X
authors.lastname: Chen
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.forename: Handong
authors.initials: H
authors.lastname: Wei
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
authors.forename: Ying
authors.initials: Y
authors.lastname: Jiang
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
mesh_terms: D000906:Antibodies; D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D018834:Chaperonin 60 / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000032:analysis*; D006515:Hepatitis B virus / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000906:Antibodies; D016227:Benzoquinones; D018834:Chaperonin 60; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; C112765:tanespimycin