Fig 1.
HBV RNA and DNA detection with specific probe in HepAD38 (DOX) cells.
HepG2-NTCP (A-B) and HepAD38 (DOX-) cells (E-L) were maintained in the doxycycline-free medium, HepAD38 (DOX+) cells (C-D) were maintained in the doxycycline medium for 7 days, then cells were fixed and stained. Prior to probe labeling, cells were treated with buffer alone (A-D, E, I), DNase I (F, J), RNase A/H (G, K), or DNase I+ RNase A/H (H, L). Probe set 2 was used for HBV (-) DNA (green); Probe set 1 was used for HBV (+) DNA and pgRNA (purple). These two probe sets were mixed and hybridized with cells and signals were captured in two independent channels (Probe set 2 Cy5, Probe set 1 Cy3), representative images were shown. Scale bar, 4 μm.
Fig 2.
Kinetics of HBV DNA and RNA accumulation during infection.
(A) HepG2-NTCP cells were infected with UV-irradiated or untreated HBV at MOI = 1000. At the indicated times post-infection, cells were fixed and processed for FISH detection. Scale bar, 4 μm. (B) Individual pgRNA and (-) DNA puncta for each time point were quantified and graphed using the R package. More than 100 cells per group were counted. (C) The median of the true pgRNA and (-) DNA signal (live virus signal subtracted by UV virus signal) over the indicated time post-infection were plotted using the smooth graph function in Microsoft Excel.
Fig 3.
Colocalization of HBV (+) DNA with H3K27ac and Pol II proteins.
HepG2-NTCP cells were infected with HBV at MOI = 1000 and at 12 days post-infection the cells were fixed and processed for viral (+) DNA detection followed by immunofluorescence staining for H3K27ac proteins with Alexa Fluor 488 labelled secondary goat anti-rabbit antibody and/or Pol II proteins with CY3 labelled secondary goat anti-mouse antibody. (A)/(C) Wide-field images. (B) Stimulated Emission Depletion (STED) super resolution images. Nuclei borders were represented by yellow dashed lines in (C). Scale bar, 4 μm. Solid white arrows point to (+) DNA colocalized with H3K27ac or with H3K27ac and Pol II proteins.
Fig 4.
Colocalization of HBV pgRNA with active translating ribosomes or capsid in HepAD38 (DOX) cell.
(A) HepAD38 (DOX-) cells were untreated or pretreated with 10 μM GLS4/Bay 41–4109 followed by puromycin (Puro) labeling. Cells were fixed and processed for pgRNA detection followed by immunofluorescence staining for puromycylated ribosomes with Alexa Fluor 488 labelled secondary goat anti-mouse antibody and capsid with Cy3 labelled secondary goat anti-rabbit antibody. Nuclei borders were represented by yellow dashed lines in the enlarged areas. Scale bar, 4 μm. (B) Voxels of capsid were quantified using Huygens. (C) Puncta of pgRNA were quantified using FISH-quant. More than 200 cells per group were counted. (D, E) Percentage of pgRNA colocalized with capsid or puromycylated ribosomes were quantified by Huygens. (F) Distance between pgRNA and capsid (Y) or puromycylated ribosomes (X) were shown as a two-dimensional plot. (G) The distribution of aforementioned X and Y values were shown as a scatter plot with marked mediums. A dashed line of 0.5 μm, the threshold for defining colocalization, was shown. ***P < 0.001 (Mann-Whitney U-test).
Table 1.
Distribution ratio of different forms of pgRNA in HepAD38 cell and in HepG2-NTCP infection system.
Fig 5.
Colocalization of HBV pgRNA with actively translating ribosomes and capsid in the HepG2-NTCP infection system.
(A) HepG2-NTCP cells were infected with HBV at MOI = 1000 and at 9 days post-infection the cells were fixed and processed for pgRNA detection followed by puromycin (Puro) labeling for puromycylated ribosomes and capsid. Colocalization of pgRNA and capsid were presented by white arrows. colocalization of pgRNA and actively translating ribosomes were presented by yellow arrows. Scale bar, 4 μm. (B) Distance between pgRNA and closest puromycylated ribosomes (X), between pgRNA and closest capsid (Y) were shown as a two-dimensional plot. (C) The distribution of aforementioned X and Y values were shown as a scatter plot with marked mediums. A dashed line of 0.5 μm, the threshold for defining colocalization, was shown.
Fig 6.
Effects of MTs disruption on HBV nucleocapsid formation.
HepAD38 cells were treated with Nocodazole or Vinblastine for 24 h before fixation and processed for HBV pgRNA (A) or (-) DNA (B), HBcAg and α-tubulin detection. White arrows indicate the pgRNA or (-) DNA were colocalized with HBcAg. Scale bar, 4 μm. (C) Analyzing pgRNA distance to its closest HBcAg within 1 μm using Huygens in FISH images. (D, E) Quantitative analysis of HBV immature and maturing nucleocapsid puncta in single cell using Huygens in FISH images. More than 15 cells per group was counted. Intracellular HBV encapsidated pgRNA (F) and nucleocapsid DNA (G) were quantified by real-time PCR. (H, I) Representative images, schematics and measurements of cellular distribution of viral (-) DNA compared to the nucleus in DMSO or MTIs-treated cells using Huygens. More than 15 cells per group was counted. NZ, Nocodazole; VIN, Vinblastine. *P < 0.05, **P < 0.01, ****P < 0.0001. ns: no significance. (C-E): Mann-Whitney U-test; (F, G): Student’s t-test, the data are representative of three independent replicates.
Fig 7.
MTs orchestrate HBV virion secretion by regulating MVBs morphogenesis.
(A) HepAD38 cells were fixed and processed for detecting HBV (-) DNA, HBsAg and MVBs specific marker CD63. Specific enlargement of the area within the white-outlined box, colocalization of (-) DNA, HBsAg and CD63 were presented by white arrows. Scale bar, 4 μm. (B) Quantification of images in DMSO-treated cells and analyzing the relationship between the mean volume of CD63+ vesicles and (-) DNA puncta in each cell. (C, D) Mean volume and puncta of CD63+ vesicles in a single cell level were measured by Huygens. Analyzing (-) DNA and CD63+ positive (E), HBsAg and CD63+ positive (F) colocalization percent per cell within 3 μm. More than 15 cells per group was counted. ****P < 0.0001 (Mann-Whitney U-test).
Fig 8.
Schematic presentation of the molecular events from cccDNA transcription to virion morphogenesis based on the observations in this study.