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.

Proteasome 19S RP and translation preinitiation complexes are secreted within exosomes upon serum starvation

“…The molecular basis of 19S RP sorting within exosomes will have to be more accurately defined, together with the fact that part of intracellular 19S RP might also be released another pathway than exosomes. Very interestingly, a study recently reported that hepatitis B virus replication modulates exosome content, in particular with an enhancement of subunits of the 19S RP It is thus tempting to speculate that HBV replication induces a cellular stress which in some way activates a signaling cascade leading to the same effect as serum starvation described here.…”

The Host-Pathogen Ecosystem Viewed Through the Prism of Proteomics

“…exosomes. This is well illustrated by Yuan and colleagues in their study of exosome composition upon infection with the important human pathogen, hepatitis B virus (10). Interestingly, exosomes contained proteasome subunits that were further shown to have the ability to regulate host immune responses.…”

Extracellular Vesicles in the Pathogenesis of Viral Infections in Humans

“…The increase in these miRNAs potentially inhibit NK cell activity via IL-12 downregulation and facilitating viral evasion of the host immune response [ 56 ]. Another study concluded through proteomic analysis of exosomes via LC-MS/MS that HBV-infected HepAD38 hepatoblastoma cell line-derived exosomes contain HBV-associated proteins capable of significantly reducing monocyte IL-6 production [ 58 ]. HBV infection in HepAD38 cells alters 35 exosome-bound proteins, including the increase of five proteasome subunit proteins: PSMD1, PSMD7, PSMD14, PSMC1, and PSMC2, enhancing proteolytic activity [ 58 ].…”

“…Another study concluded through proteomic analysis of exosomes via LC-MS/MS that HBV-infected HepAD38 hepatoblastoma cell line-derived exosomes contain HBV-associated proteins capable of significantly reducing monocyte IL-6 production [ 58 ]. HBV infection in HepAD38 cells alters 35 exosome-bound proteins, including the increase of five proteasome subunit proteins: PSMD1, PSMD7, PSMD14, PSMC1, and PSMC2, enhancing proteolytic activity [ 58 ]. Inhibition of exosome-dependent proteasomal activity resulted in increased IL-6 production, implying proinflammatory molecules modulated by proteasomal subunit proteins within HepAD38 exosome transport [ 58 ].…”