Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction

Abstract

Evidence suggests that exosomes can transfer genetic material between cells. However, their roles in hepatitis B virus (HBV) infection remain unclear. Here, we report that exosomes present in the sera of chronic hepatitis B (CHB) patients contained both HBV nucleic acids and HBV proteins, and transferred HBV to hepatocytes in an active manner. Notably, HBV nucleic acids were detected in natural killer (NK) cells from both CHB patients and healthy donors after exposure to HBV-positive exosomes. Through real-time fluorescence microscopy and flow cytometry, 1,1'-dioctadecyl-3,3,3',3',-tetramethylindodicarbocyanine, 4-chlorobenzenesulfnate salt (DiD)-labeled exosomes were observed to interact with NK cells and to be taken up by NK cells, which was enhanced by transforming growth factor-β treatment. Furthermore, HBV-positive exosomes impaired NK-cell functions, including interferon (IFN)-γ production, cytolytic activity, NK-cell proliferation and survival, as well as the responsiveness of the cells to poly (I:C) stimulation. HBV infection suppressed the expression of pattern-recognition receptors, especially retinoic acid inducible gene I (RIG-I), on NK cells, resulting in the dampening of the nuclear factor κB(NF-κB) and p38 mitogen-activated protein kinase pathways. Our results highlight a previously unappreciated role of exosomes in HBV transmission and NK-cell dysfunction during CHB infection.

Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy

“…Indeed, the Cys residues within the S domain form several disulfide bonds that confer stability to the virions and might also be essential for the virion uptake and productive infection [34]. Although we did not examine the infectious ability of the isolates with sC69*, infection of new hepatocytes through alternative secretion of the isolates, as evidenced via exosomes in our study, is expected even if the incomplete structure of envelop proteins does not support the infectivity [15]. …”

“…HBV progeny DNA was extracted subsequently through alcohol precipitation. To isolate HBV DNA from secreted particles, three different approaches were applied: (a) polyethylene glycol precipitation (PEG, Sigma, St. Louis, MO, USA), followed by capsid digestion and alcohol precipitation of DNA; (b) immune precipitation of released particles by using preS1 (Santa Cruz Biotechnology, USA) and core (Thermo Fisher, USA) antibodies and isolation of the DNA (QIAamp DNA mini kit, Qiagen, USA); (c) ultra-centrifugation of the supernatants (110,000x g, 70 min) [15], evaluation of the concentration of pelleted particles using the LM10 nanoparticle characterization system in real-time (NanoSight, Malvern Instruments) equipped with a blue laser (405 nm) followed by isolation of exosomes from precipitated particles by using an Exosome-Human CD63 isolation/detection reagent (Thermo Fisher) and extraction of DNA (QIAamp DNA mini kit). A 5 μl aliquot of HBV DNA isolated from immunoprecipitated particles (using preS1 and core antibodies) and exosomes was subjected per reaction well to qPCR analysis, using the iTaq Universal SYBR Green One-Step qPCR kit (BioRad, Hercules, CA).…”

Decoding the role of extracellular vesicles in liver diseases

“…76 Moreover, exosomes isolated from sera of chronic hepatitis B patients contain HBV viral components and can induce active infection in naive human hepatocytes. 77 Recently, Yang et al 77 found that exosomes shuttle HBV viral components into NK cells, leading to NK cell dysfunction through the downregulation of retinoic acid inducible gene I (RIGI) and inactivation of nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinase pathways.…”

Extracellular vesicles from hepatitis B patients serve as reservoir of hepatitis B virus DNA

“…This may be used as a marker for identifying potential reactivation candidates, which needs to be validated and further longitudinal studies should be performed. The EVs carrying HBV transmits infection are already described previously by Yang et al in hepatoma cell lines. The novelty of our study is that it highlights the detection of HBV DNA in EVs when it was undetectable in plasma of same sample.…”