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.
Highlights
Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide
The most intensely stained protein spot that was isolated from the 450-kDa complex on the two-dimensional gel (Spot 8) was shown to have an electrophoretic mobility of 60 kDa. This protein was identified as HSP60, a molecular chaperone that has been shown to be associated with HBV replication by interacting with and activating HBV polymerase (HBV pol) [17]
We utilized blue native (BN)-PAGE to analyze differences in protein complexes present in HepG2 and HepG2.2.15 cell lysates to identify cellular proteins involved in the HBV life cycle
Summary
Cell Culture—HepG2 and HepG2.2.15 were grown in minimal Eagle’s medium (Hyclone) supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin/streptomycin, and 2% L-glutamine at 37 °C and 5% CO2 in a humidified atmosphere. For the antibody-based gel supershift assays, approximate 80 g of HepG2.2.15 cellular protein extracts were combined with 1 g of anti-HSP60 antibody (Santa Cruz Biotechnology) and incubated on ice for 30 min before loading the mixture on the BN-PAGE. An equivalent concentration of HepG2.2.15 cellular protein lysates was combined with 1 g of an irrespective antibody (normal IgG; Santa Cruz Biotechnology) used as a negative control. After in-gel digestion with trypsin, the extracted peptide mixtures were loaded onto a nanoscale LC-ESI-Q-TOF MS instrument (Q-TOF Micromass spectrometer, Waters) for protein identification as described elsewhere [15]. The immune complexes were precipitated with protein A/G-agarose (Santa Cruz Biotechnology) for 2 h at 4 °C, the samples were washed, proteins were separated by SDS-PAGE, and expression was determined by immunoblot analysis. The above experiments were performed in triplicate, and the results are presented as the mean Ϯ S.D
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