Presence of Intact Hepatitis B Virions in Exosomes

Abstract

Background & aims: Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome biogenesis. In light of this, it was studied whether intact virions wrapped in exosomes are released by HBV-producing cells.

Methods: Robust methods for efficient separation of exosomes from virions were established. Exosomes were subjected to limited detergent treatment for release of viral particles. Electron microscopy of immunogold labeled ultrathin sections of purified exosomes was performed for characterization of exosomal HBV. Exosome formation/release was affected by inhibitors or Crispr/Cas-mediated gene silencing. Infectivity/uptake of exosomal HBV was investigated in susceptible and non-susceptible cells.

Results: Exosomes could be isolated from supernatants of HBV-producing cells, which are characterized by the presence of exosomal and HBV markers. These exosomal fractions could be separated from the fractions containing free virions. Limited detergent treatment of exosomes causes stepwise release of intact HBV virions and naked capsids. Inhibition of exosome morphogenesis impairs the release of exosome-wrapped HBV. Electron microscopy confirmed the presence of intact virions in exosomes. Moreover, the presence of large hepatitis B virus surface antigen on the surface of exosomes derived from HBV expressing cells was observed, which conferred exosome-encapsulated HBV initiating infection in susceptible cells in a , large hepatitis B virus surface antigen/Na⁺-taurocholate co-transporting polypeptide-dependent manner. The uptake of exosomal HBV with low efficiency was also observed in non-permissive cells.

Conclusion: These data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV.

Keywords: MVB; Ultrathin Cryosection; Virion Egress; Virus-host Interaction.

Figure 1

Isolation and characterization of exosomes purified from human hepatoma cell lines.A, Schematic description of the exosome purification procedure from the culture fluids of human hepatoma cell lines. B, The size distribution of exosomes isolated from HBV-negative HepG2 cells and from the HBV-expressing stable cell line HepAD38. Analysis of size distribution was performed by NanoSight NS300. The error bars were shown as dotted lines with fill areas. C, Exosomes were isolated from the supernatant of HepAD38 (left) or HepG2 cells (right) as shown in Figure 1, A by differential centrifugation followed by iodixanol density gradient centrifugation. The fractions of the iodixanol gradient were analyzed by Western blot detecting Alix, Tsg101, and CD63 as exosomal markers and LHBs and HBcAg as HBV components. D, Absolute quantification of the distribution of HBV genomes (black line) and of HBcAg (quantification of the Western blot in Figure 1, C) (blue dotted line) in each fraction in the iodixanol gradient. The density is shown by the red line. E, Quantification of HBsAg in each fraction in the iodixanol gradient by ELISA. The fractions were either left untreated (red bars) or pretreated with RIPA buffer and ultrasound sonication (blue bars). Two-way analysis of variance followed by the Sidak multiple comparison test for all panels. ∗∗∗P < .001. F, IP assay of the exosomal iodixanol gradient fractions with CD63-coated magnetic beads or unrelated Dynabeads M-280 Sheep Anti–Rabbit IgG as control. The input, unbound supernatants, and immunoprecipitated targets from the fractions were analyzed by Western blot, detecting Alix as exosomal marker and LHBs and HBcAg as HBV components. G, The percentage of exosome-associated HBV genomic DNA and exosomal-associated HBcAg as compared with the total input supernatant. HBV genomic DNA in the exosomal fractions and in the input of the gradient was quantified by qPCR. Quantification of HBcAg was performed by HBcAg-specific ELISA.

Figure 2

Separation of exosomal HBV and free HBV by iodixanol- and sucrose-density centrifugation.A, Immunogold labeling of exosomal fractions from the iodixanol gradient as shown in Figure 1, A was visualized by phosphotungstic acid negative staining with an anti-CD63 antibody (10 nm gold). B, Immunogold phosphotungstic acid staining the HBV virions fraction from the iodixanol gradient as shown in Figure 1, A using an anti-preS1/preS2 domain rabbit serum (K112-4, 10 nm gold). C, Schematic description: exosomal fractions 7 to 9 or free HBV virions fractions of the iodixanol gradient were pooled. One-half was left untreated; the other half was adjusted to 0.5% NP-40 and incubated for 2 hours at 37 ^oC. Subsequently, re-centrifugation on an iodixanol or sucrose density gradient was performed. D, Absolute quantification of the distribution of HBV genomes by qPCR in each fraction in the iodixanol gradient. The untreated input is represented by the black line. The NP-40-treated input is represented by the red line. The density is shown by the gray line. E, Western blot analysis of the fractions of the iodixanol gradient centrifugation of the untreated input and of the NP-40-treated input. For detection, the LHBs- and core-specific antibodies and the Alix-specific antibody were instrumental. F, Quantification of HBcAg was performed by HBcAg-specific ELISA in fraction 8 (NP-40-untreated input) and fractions 11, 12 (NP-40-treated input) of the iodixanol gradients (Figure 2, D) in the presence or absence of Triton X-100 detergent. Unpaired parametric t tests for all panels; ∗∗P < .01.

Figure 3

Separation of free HBV virions and naked capsids from exosomal HBV.A, Gradient purified free HBV virions (see schematic representation in Figure 2, C) with or without prior treatment with 0.5 % NP-40 for 40 minutes at 37 °C were being subjected to iodixanol gradient re-centrifugation. Copy number of HBV genomes in gradient fractions was quantified by qPCR. The black line shows the distribution of the untreated input; the red line is the NP-40 treated input. The density is shown by the gray line. B, Gradient purified free HBV virions treatment with 0.5 % NP-40 for 40 minutes at 37 °C (red line) or without prior treatment with NP-40 (black line) were being subjected to sucrose density gradient centrifugation. Copy number of HBV genomes in gradient fractions was quantified by qPCR. The density is shown by the gray line. C, Quantification of core antigen by ELISA in fraction 12 (NP-40-untreated) and fraction 15 (NP-40-treated) of the sucrose gradient (Figure 3, B) without and with Triton X-100 pretreatment. Unpaired parametric t tests for all panels; ∗∗∗P < .001. D, Sucrose density gradient of iodixanol gradient purified exosomes after pretreatment without (black line) or with 0.25 % NP-40 (red line) for 20 minutes at 30 °C. Copy number of HBV genomes in gradient fractions was quantified by qPCR. The density is shown by the gray line. E, HBV virions were visualized by phosphotungstic acid negative staining of fractions 11 and 12 (NP-40-treated) of the sucrose gradient in Figure 3D. The arrows highlight the virus. F, Western blot analysis of the fractions of the sucrose gradient centrifugation of the untreated input and of the NP-40-treated input shown in Figure 3, D. For detection, the LHBs- and core-specific antibodies and the Alix-specific antibody were instrumental. G, Quantification of core antigen by HBcAg-specific ELISA in fractions 8, 11, 12, and 14, 15 of the sucrose gradients (Figure 3, D) without and with Triton X-100 pretreatment. Unpaired parametric t tests for all panels; ∗P < .05; ∗∗∗P < .001. H, Sucrose density gradient of iodixanol gradient purified exosomes after pretreatment without (black line) or with 0.5 % NP-40 (red line) for 1 hour at 37 °C. Copy number of HBV genomes in gradient fractions was quantified by qPCR and shown in the upper panel. The density is shown by the gray line. The HBcAg distribution over the gradient of 0.5 % NP-40-treated input was analyzed by Western blot and shown in the lower panel. I, Phosphotungstic acid stain followed by TEM imaging of naked capsids detected in fractions 14, 15 (NP-40-treated) of the sucrose gradient shown in Figure 3, H or detected in fraction 15 (NP-40-treated) of the sucrose gradient shown in Figure 3, D. Arrows highlight the naked capsids.

Figure 4

Inhibition of MVB- or exosome-formation impairs release of membrane-cloaked HBV virions.A, HBV genome distributions in iodixanol density gradient of exosomes purified from the supernatant of HepAD38 cells. Cells were treated with 2 μg/mL (red dotted line) or 4 μg/mL U18666A (blue dotted line) for 48 hours or left untreated (black line). Viral genomes were quantified by qPCR. B, Western blot analysis of the fractions of the iodixanol gradient centrifugation (Figure 4, A) of the input derived from untreated cells (upper panel) or with U18666A-treated cells (lower panel). For detection, Alix- and Tsg101-specific antibodies- and core-specific antibodies were instrumental. C, HBV genome distributions in iodixanol density gradient of exosomes purified from the supernatant of HepAD38 cells treated with 10 μM (red dotted line) or 15 μM of manumycin A (blue dotted line) for 48 hours or untreated cells (black line). The viral genomes were quantified by qPCR. D, HBV genome distributions in iodixanol density gradient of exosomes purified from the supernatant of HepAD38 cells treated with 25 μM (red dotted line) or 50 μM GW4869 (blue dotted line) for 48 hours or untreated cells (black line). The viral genomes were quantified by qPCR. E, Alix distributions in an iodixanol-based density gradient (Figure 4, C–D) of exosomes purified from the supernatant of HepAD38 cells treated with manumycin A (left panel) or GW4869 (right panel) for 48 hours or untreated cells. F, Representative Western blot of the intracellular LHBs and HBcAg from HepAD38 cells treated with U18666A (left panel) or manumycin A (middle panel) or GW4869 (right panel) for 48 hours or from untreated cells. Detection of β-actin served as loading control. G, Quantification of intracellular LHBs (upper panel) and HBcAg (lower panel) signals in Figure 4, F. Fold change compared with untreated cells. Unpaired parametric t tests for all panels. H, HBV total RNAs expression levels from HepAD38 cells treated with U18666A or manumycin A or GW4869 for 48 hours or untreated cells. HBV total RNA were quantified by reverse transcription qPCR, and the relative expression levels to RPL27 mRNA were plotted as fold change to untreated cells. Unpaired parametric t tests for all panels. I, The extracellular levels of HBsAg (upper panel) and HBeAg (lower panel) in U18666A-, manumycin A-, or GW4869-treated HepAD38 cells were measured by ELISA. The fold change with untreated cells was plotted. Unpaired parametric t tests for all panels; ∗∗P < .01.

Figure 5

CRISPR/Cas9 mediated knockout of Alix or Syntenin in HepAD38 cells impairs the release of exosomal HBV virions.A–B, Western blot analysis of cellular lysates derived from Alix-deficient HepAD38 cells (A) or Syntenin-deficient HepAD38 cells (B). Detection of β-actin served as loading control. C, Intracellular amount of LHBs and HBcAg from Alix- or Syntenin-deficient HepAD38 cells. Representative Western blots for LHBs, HBcAg, and β-actin are shown in the left panel. Quantification of LHBs and HBcAg signals are shown in the right panel; fold change compared with NT cells. Unpaired parametric t tests for all panels; ∗∗P < .01. D, HBV total RNAs expression levels from Alix- or Syntenin-deficient HepAD38 cells. HBV total RNA were quantified by reverse transcription qPCR, and the relative expression levels to RPL27 mRNA were plotted as fold change to NT cells. Unpaired parametric t tests for all panels. E, The amount of extracellular HBV DNA from NT cells or Alix- or Syntenin-deficient HepAD38 cells was evaluated by qPCR. Unpaired parametric t tests for all panels. F–G, Extracellular HBsAg levels (F) and extracellular HBeAg levels (G) in Alix- or Syntenin-deficient HepAD38 cells were measured by ELISA. Fold change compared with NT cells were plotted. Unpaired parametric t tests for all panels. H–I, HBV genome distributions in iodixanol density gradient of exosomes purified from the supernatant of HepAD38 NT cells (black line), of Alix-deficient HepAD38 cells (blue dotted line), and of Alix-deficient HepAD38 cells rescued by overexpression of mCherry-Alix (red dotted line). The viral genomes were quantified by qPCR. The absolute quantification was displayed in Figure 5, H. Fold change of each gradient to fraction 12 was shown in Figure 5, I. J, Western blot analysis of the fractions of the iodixanol gradient centrifugation (Figure 5, H–I) of the input derived from HepAD38 NT cells (upper panel) or from Alix-deficient HepAD38 cells (middle panel) or from Alix-deficient HepAD38 cells rescued by overexpression of mCherry-hAlix (lower panel). For detection, the Alix-specific and the core-specific antibodies were instrumental. K–L, HBV genome distributions in iodixanol density gradient of exosomes purified from the supernatant of HepAD38 NT cells (black line) or of Syntenin-deficient HepAD38 cells (blue dotted line). The viral genomes were quantified by qPCR. The absolute quantification is displayed in Figure 5, K. Fold change of each gradient to fraction 12 is shown in Figure 5, L.

Figure 6

Transmission electron microscopy of exosomes released from HepAD38 cells.A, TEM images from ultra-thin sections of Epon-embedded (left) or cryo-sections (right) of fixed exosomes showing virions enclosed by a membrane structure (labeled by arrows). An asterisk indicates that a dense viral envelope stands out from the surrounded nucleocapsid. B–C, Immunogold labeling of ultra-thin thawed cryo-sections of fixed exosomes. The cryo-sections were either labeled with an anti-CD63 antibody (visualized by 10-nm gold particles) or an anti LHBs antiserum (anti-preS1/preS2 domain rabbit serum [K112-4]) (visualized by 5-nm gold particles). Arrows indicate specific colloidal gold labeling. Asterisks represent that anti-LHBs (5 nm) is located at identifiable enclosed virus like particles. The surface of cryo-sectioned exosomes was also labeled by anti-LHBs (orange arrows).

Figure 7

Inoculation of HepG2- and differentiated HepaRG cells by exosomal HBV and free HBV.A, HBV genome copy number in each exosomal and viral inoculum is shown in the left. Schematic description of the infection experiment procedure is shown in the right. B, HBsAg ELISA of supernatant derived from HepG2 cells inoculated either with exosomal HBV virions or free HBV virions. Medium was changed at the indicated time points and analyzed by HBsAg ELISA. The horizontal dotted line indicated the cutoff value. C, HBsAg ELISA of supernatant derived from differentiated HepaRG cells inoculated either with exosomal HBV (fraction 8 of the iodixanol gradient) or a less pure, later exosomal fraction (fraction 10). The inoculum was preincubated with the LHBs-specific antibody MA18/7 (1 μg/mL) or an anti-hexa-His-specific monoclonal antibody (1 μg/mL) as control. Medium was changed at the indicated time points and analyzed by HBsAg-specific ELISA. S/CO, Sample to cutoff signal. The cutoff value is represented by the horizontal dotted line. D, HBsAg ELISA of supernatant derived from differentiated HepaRG cells inoculated with free HBV virions (fractions 13 and 14). The inoculum was preincubated with the LHBs-specific antibody MA18/7 (1 μg/mL) or an anti-hexa-His-specific monoclonal (1 μg/mL) as control. Medium was changed at the indicated time points and analyzed by HBsAg-specific ELISA. The horizontal dotted line delineates the cutoff value. E, HBsAg ELISA of supernatant derived from differentiated HepaRG cells inoculated either with exosomal HBV (fractions 7, 8 of the iodixanol gradient) or a less pure, later exosomal fraction (fractions 9, 10 of the iodixanol gradient). The inoculum was preincubated with the LHBs-specific antibody MA18/7 (1 μg/mL) or an anti-hexa-His-specific monoclonal (1 μg/mL) as control. Here, the HBsAg content at day 4 of the different samples is analyzed. Unpaired parametric t tests for all panels; ∗∗P < .01; ∗∗∗P < .001; ns, not significant. F–G, Differentiated HepaRG cells were inoculated with free HBV virions (fractions 13) (F) or exosomal HBV (fraction 8 of the iodixanol gradient) (G), and secreted HBsAg was quantified at the indicated time points by HBsAg-specific ELISA. Myrcludex B 500 nM was added during infection. The horizontal dotted line indicates the cutoff value.

Figure 8

Inoculation of HepG2-NTCP and HepG2 cells by exosomal HBV isolated from the supernatant of HepAD38 cells.A, Representative immunofluorescence staining of HBsAg (green) on day 7 of HepG2-NTCP cells inoculated with or without 2-fold amounts of the purified exosomes used in Figure 7; nuclei in blue, scale bar = 15.1 μm. B, Representative immunofluorescence staining of HBsAg (green) on day 7 of HepG2 cells inoculated with or without 2-fold amounts of the purified exosomes used in Figure 7; nuclei in blue, scale bar = 15.1 μm. C, Two-fold amounts of the purified exosomes used in Figure 7 were inoculated with HepG2 and HepG2-NTCP cells, and HBeAg in the supernatant of these cells was quantified by HBeAg-specific ELISA at the indicated time points. The horizontal dashed line represents the cutoff value.