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.

Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

“…Flag or HA tags were introduced in some constructs to enable easier detection of expressed protein. Plasmid carrying terminally redundant 1.1X copy HBV genome downstream of CMV promoter with the HBx ORF nulled by introducing premature termination at residue 8 (1.1HBV-X − ) has been previously described10. CMV-driven eGFP expression plasmid (Beyotime) was used for transfection efficiency control.…”

“…Exosomes were prepared from cell culture supernatants using the classical method of differential centrifugation essentially as previously reported10. Briefly, supernatants were first centrifuged at 300  g for 10 min to remove detached cells and cell debris was removed by centrifugation at 2,000  g for 15 min followed by centrifugation at 10,000  g for 30 min.…”

“…HBx is not considered to be a viral structural protein and numerous studies over the decades have shown that HBx plays a myriad of functions in regulating viral life cycle, host-virus interactions, as well as HBV-related HCC5678. Most notably, HBx acts to stimulate the activities of HBV promoters and enhancers: obliteration of functional HBx ORF results in prominently reduced viral transcription and replication in vitro , which could be rescued by supplementing HBx expression in trans 910. Infection experiments in vivo further confirms that HBx is essential for initiating and maintaining HBV life cycle7.…”

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells

“…By applying an SILAC quantitative proteomics approach, Zhao and colleagues revealed that hepatitis B virus also alters the proteomic composition of hepatic cell line Huh-7-derived EVs [58]. A similar modulation was observed in the case of human T cell lymphotropic virus-infected T cell lines [59].…”

Hepatitis B virus X protein in liver tumor microenvironment

“…Negatively regulating the expression of exosomal miR-122In vitro studies of transfected cell lines HepG2 and Huh-7 cellSong et al [36]2. Significantly altering the exosomal protein contentIn vitro studies of transfected cell lines Huh-7 cellZhao et al [37] …”