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.
Highlights
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; §Medizinisches Proteom-Center, Ruhr-Universitat Bochum, 44801 Bochum, Germany
Employing unbiased label-free LC-Mass spectrometry (MS)/MS-based quantification analyses of exosomal proteins, we demonstrated that exosomes secreted from HepAD38 cell line, which supports Hepatitis B virus (HBV) gene replication, are packed with proteins associated with HBV that can modulate production of IL-6 by monocytes
We used proteomics approaches to evaluate the differences in protein content of exosomes secreted by the HepAD38 cell line with or without HBV replication. 1412 exosome protein groups were identified, including proteins involved in vesicle-mediated transport, establishment of protein location, immune effector related process as well as proteasome pathway
Summary
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; §Medizinisches Proteom-Center, Ruhr-Universitat Bochum, 44801 Bochum, Germany. 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, providing a new perspective on the role of exosomes in HBV-host interaction. The main goal of this study was to get insights into how the HBV gene replication modulates the protein content of exosomes secreted from hepatocytes
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