Journal: mSphere

Article Title: Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome

doi: 10.1128/msphere.00518-24

Figure Lengend Snippet: Detection of HBcAg and HiBiT-tagged viral proteins by transfection of HiBiT-tagged HBVcc plasmids. ( A ) The production of HBcAg and HiBiT-tagged viral proteins was detected by immunostaining with anti-HBc and anti-HiBiT antibodies 3 days after transfection. Nuclei were visualized by staining with DAPI. The white bar indicates 100 µM. ( B ) The HiBiT signals in cells and culture medium were measured after transfection of plasmids for HiBiT-tagged HBVcc. HBVcc-WT was used as a negative control. ( C ) HBsAg production in the culture medium was measured after transfection of plasmids for HiBiT-tagged HBVcc.

Article Snippet: The expression of HBcAg in transfected cells was detected by staining with a rabbit polyclonal anti-HBc antibody (Beacle Inc., Kyoto, Japan) and Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Transfection, Immunostaining, Staining, Negative Control

Journal: mSphere

Article Title: Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome

doi: 10.1128/msphere.00518-24

Figure Lengend Snippet: Infection of HiBiT-tagged HBVcc in human primary hepatocytes. ( A ) Human primary hepatocytes were infected with HiBiT-tagged HBVcc at 200 GEq/cell, and the infection efficiencies were compared with those of HBVcc-WT by monitoring HBsAg and HiBiT signals in culture medium on the indicated days after infection. ( B ) The HBV DNA levels in the culture media of HBVcc-WT- and HiBiT-tagged HBVcc-infected cells were assessed by real-time PCR with a primer and probe set designed to target the HBs region after treatment with DNase after 12 days of culture. ( C ) HBVcc-WT- and HiBiT-tagged HBVcc-infected cells were detected by staining with rabbit polyclonal anti-HBc antibody and Alexa Fluor 555-conjugated anti-rabbit IgG. Nuclei were visualized by staining with DAPI.

Article Snippet: The expression of HBcAg in transfected cells was detected by staining with a rabbit polyclonal anti-HBc antibody (Beacle Inc., Kyoto, Japan) and Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Infection, Real-time Polymerase Chain Reaction, Staining

Journal: mSphere

Article Title: Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome

doi: 10.1128/msphere.00518-24

Figure Lengend Snippet: Infection of HepG2/NTCP cells with HiBiT-tagged HBVcc.( A ) HepG2/NTCP cells were infected with HiBiT-tagged HBVcc at 200 GEq/cell, and the infection efficiencies were compared with those of HBVcc-WT by monitoring HBsAg and HiBiT signals in the culture media on the indicated days after infection. ( B ) The HBV DNA levels in the culture media of HBVcc-WT- and HiBiT-tagged HBVcc-infected cells were assessed by real-time PCR with a primer and probe set designed to target the HBs region after treatment with DNase after 12 days of culture. ( C ) HBVcc-WT- and HiBiT-tagged HBVcc-infected cells were detected by staining with rabbit polyclonal anti-HBc antibody and Alexa Fluor 555-conjugated anti-rabbit IgG. Nuclei were visualized by staining with DAPI.

Article Snippet: The expression of HBcAg in transfected cells was detected by staining with a rabbit polyclonal anti-HBc antibody (Beacle Inc., Kyoto, Japan) and Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Infection, Real-time Polymerase Chain Reaction, Staining

Journal: mBio

Article Title: Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division

doi: 10.1128/mbio.03303-22

Figure Lengend Snippet: Changes to the subcellular distribution of HBV Cp over time. (A) Cartoon illustration of Cp divided into the N-terminal Assembly Domain and C-Terminal Domain (CTD). The CTD encodes the nuclear localization signal (NLS). The HBV Cp dimer crystal structure shown is based on PBD: 3J2V . The position of the Cp-Y132A mutation is highlighted in blue, shown on the red Cp subunit. (B) Outline of HBV expression and time course analysis workflow based on immunofluorescence (IF) detection of WT HBV Cp or Cp-Y132A. Images were developed using BioRender. (C) Representative images and quantification from IF time-course analysis of Huh7 cells expressing WT Cp with packageable pgRNAs, or the Cp-Y132 (no assembly) control. Cells were fixed at the indicated time points with WT Cp or Cp-Y132A detected using polyclonal anti-HBc antiserum. White dashed lines differentiate nuclei (N) from cytoplasm (C). Red dashed boxes highlight regions of interest, with red arrows indicating WT Cp puncta consistent with assembled capsids. Image scale bars represent 10 μm. Plots on the right present ratios of C/N mean fluorescence intensity (MFI) for 100 cells per condition per time point. The red dashed line at 1 indicates equivalent levels of nuclear and cytoplasmic fluorescence signals. Greater than 1 indicates more cytoplasmic MFI relative to the nucleus. Less than 1 indicates more nuclear MFI relative to the cytoplasm.

Article Snippet: For global Cp detection, a 1:1000 dilution of a polyclonal rabbit anti-core (anti-HBc) antiserum (Lot#:214-14, Austral Biologicals) was used prior to washing with PBS and staining with 1:1000 diluted secondary antibodies (goat anti-rabbit 488; Life Technologies).

Techniques: Mutagenesis, Expressing, Immunofluorescence, Fluorescence

Journal: mBio

Article Title: Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division

doi: 10.1128/mbio.03303-22

Figure Lengend Snippet: Cp forms high-order assemblages in the nucleus. (A) Illustrations of Cp dimer and capsid structures highlighting the HBV Cp binding sites for the mAb3120 and polyclonal (anti-HBc) antibodies used for the dual labeling strategy. (B) Representative images from IF analysis of cells expressing WT Cp or Cp-Y132A, and incubated with the indicated antibodies, to confirm binding specificity. mCherry (inset, red) was co-transfected with the Cp variant to assist in identifying transfected cells prior to fixation, staining with DAPI (blue), and staining for IF (green). mAb3120 was confirmed as unable to detect Cp-Y132A (central panels). (C) Images and analysis of dually labeled Huh7 cells expressing WT Cp and detected using mAb3120 (cyan) and polyclonal anti-HBc (green). Transect analysis illustrates differential detection of Cp with polyclonal (predominantly nuclear) and mAb3120 (predominantly cytoplasmic) at 72h in a representative cell; consistent with unassembled Cp trafficking to the nucleus, even when the assembled capsid population is predominantly in the cytoplasm. Scale bars represent 10 μm. (D) Bar graphs quantifying the subcellular localization of WT Cp for 100 cells per condition, detected using the indicated antibody and corresponding to the experiment in (C). Error bars represent the standard deviation of the mean for 3 biological replicates. (E) Bar graphs as for (D) confirming a similar subcellular localization of WT Cp distribution over time when expressed from a plasmid encoding envelope glycoproteins (Env+).

Article Snippet: For global Cp detection, a 1:1000 dilution of a polyclonal rabbit anti-core (anti-HBc) antiserum (Lot#:214-14, Austral Biologicals) was used prior to washing with PBS and staining with 1:1000 diluted secondary antibodies (goat anti-rabbit 488; Life Technologies).

Techniques: Binding Assay, Labeling, IF-cells, Incubation, Transfection, Variant Assay, Staining, Expressing, Standard Deviation, Plasmid Preparation

Journal: mBio

Article Title: Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division

doi: 10.1128/mbio.03303-22

Figure Lengend Snippet: Inducing cell cycle arrest using aphidicolin that entraps HBV Cp/capsids in the nucleus. Representative images showing Huh7 cells expressing WT HBV, treated with 10 μg/mL APC for the indicated time period (e.g., 24, 48, and 72 h post-gene expression). In the presence of APC, WT Cp remained predominantly nuclear at all time points, as detected by both capsid-specific mAb3120 and anti-HBc polyclonal antibody. Smaller images (top) bordered in red show WT HBV control WT Cp expression and relocalization from the nucleus to the cytoplasm in the absence of APC. Scale bars represent 10 μm. Bar graphs present data from 3 independent experiments, measuring 100 cells per condition and time point, with error bars representing the standard deviation of the mean.

Article Snippet: For global Cp detection, a 1:1000 dilution of a polyclonal rabbit anti-core (anti-HBc) antiserum (Lot#:214-14, Austral Biologicals) was used prior to washing with PBS and staining with 1:1000 diluted secondary antibodies (goat anti-rabbit 488; Life Technologies).

Techniques: Expressing, Standard Deviation

Journal: mBio

Article Title: Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division

doi: 10.1128/mbio.03303-22

Figure Lengend Snippet: Increasing rates of capsid assembly does not affect Cp’s preferential accumulation in the nucleus at early time points. (A) Representative images of cells expressing rapid assembly mutant Cp-V124W tracked over a 72 h time course using dual label IF analysis. The red arrows indicate cytoplasmic co-localization of capsid/Cp detected by both mAb3120 and polyclonal anti-HBc antisera, respectively. Graphs present relative levels of nuclear versus cytoplasmic Cp-V124W distribution for 100 cells per time point. Error bars represent the standard deviation of the mean for 3 independent experiments. (B) Images from live single cell detection of fluorescent Cp-V124W-NG co-expressed with untagged Cp-V124W over an ~70 h time course. Time point labeled in red (28.5h) designates a cell division event. Please reference for the complete video file. Graph shows tracking of nuclear and cytoplasmic Cp-V124W-NG MFI for a representative cell, plotted over time. Gray box highlights an ~6 h time window encompassing a cell division event. (C) Images from IF detection of untagged WT Cp and Cp-V124W using polyclonal anti-HBc (green) with cellular nucleoli labeled using anti-Nucleophosmin (B23) (magenta) antibodies, indicating that both WT Cp and Cp-V124W localize to the nucleolus at early time points. Scale bars represent 10 μm. Red arrows highlight co-incident detection of Cp or Cp-V124W with Nucleophosmin (B23).

Article Snippet: For global Cp detection, a 1:1000 dilution of a polyclonal rabbit anti-core (anti-HBc) antiserum (Lot#:214-14, Austral Biologicals) was used prior to washing with PBS and staining with 1:1000 diluted secondary antibodies (goat anti-rabbit 488; Life Technologies).

Techniques: Expressing, Mutagenesis, Standard Deviation, Labeling

Journal: PLoS ONE

Article Title: C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich 167 RRRSQSPRR 175 Domain Is Critical for HBV Replication

doi: 10.1371/journal.pone.0041087

Figure Lengend Snippet: (A) Schematic diagrams of HBV, DHBV, and chimeric C protein variant constructs aligned with amino acid sequences of HBV and DHBV C protein carboxyl-terminal domains. Amino acids in bold are identical or homologous. SRPK and PKA phosphorylation sites of HBV are marked with asterisks and arrowheads, respectively. Phosphorylation sites of DHBV , are marked with open arrowheads. Amino acid sequences of the HBV and DHBV C proteins are presented as open and closed boxes, respectively. The cytomegalovirus immediate early (CMV IE) promoter is represented as an open arrow. PRE, post-transcriptional regulatory element. (B) Identification of C protein and core particles by chimeric C protein variants. To examine expression of C protein variants, lysates from HuH7 cells transfected with a pHCP, pDCP, pHD192–262, pHD192–220, pHD221–262, pHCP145, pHCP145–R127Q, or C-deficient mutant were electrophoresed on 12% SDS-PAGE gels and protein levels visualized by Western blotting using polyclonal rabbit anti-HBc antibody (top panel). C protein variants (arrowheads) with expected molecular weights are indicated. The C-deficient mutant lacks C protein due to the introduction of a stop codon at Glu 8 in the C ORF. The pHCP and the C-deficient mutant constituted positive and negative controls, respectively. Transfection experiments were repeated four times. To detect core particles formed by C protein variants from native agarose gels, isolated core particles were transferred to PVDF membranes and incubated with polyclonal rabbit anti-HBc antibody (second panel). The Renilla luciferase expression plasmid phRL-CMV was co-transfected into HuH7 cells as a transfection control (third panel). Luciferase and α-tubulin (bottom panel) levels were determined by Western blotting using polyclonal rabbit anti-luciferase and monoclonal mouse anti-tubulin antibodies as transfection and loading controls, respectively. HRP-conjugated secondary antibody and enhanced chemiluminescence were used to visualize C, α-tubulin, and luciferase proteins and core particles. (C) Relative levels of C protein expression and core particle assembly by chimeric C protein variants. Relative levels of C proteins, core particles, and luciferase were measured with the Fujifilm Image Gauge V4.0 program. Relative levels of C protein variant expression and core particle assembly were compared to normalized transfection efficiencies. The data represent the mean ± standard deviation (SD) from four independent experiments.

Article Snippet: For detection of core particles, both polyclonal rabbit anti-HBc antibody (diluted 1∶1000; DAKO, Carpinteria, CA, USA) and our antibody (diluted 1∶1000) were used interchangeably ( ).

Techniques: Variant Assay, Construct, Expressing, Transfection, Mutagenesis, SDS Page, Western Blot, Isolation, Incubation, Luciferase, Plasmid Preparation, Standard Deviation

Journal: Virus Research

Article Title: Identification of neutralizing epitopes in the preS2 domain of the hepatitis B virus

doi: 10.1016/j.virusres.2022.199014

Figure Lengend Snippet: Immunization of mice with plasmids encoding HBV preS1 and preS2. (A) Schematic diagram of the structure of HBsAg and of plasmids encoding N-terminal preS1 and preS2, showing the position of the CAG promoter (CAG) and of sequences encoding the murine Ig κ-chain leader sequence (S), Myc tag (myc), spacer, and GroEL. The amino acid sequences of preS1/2–47 and preS2 are derived from HBV genotype-C. (B) Expression of preS1/2–47 and preS2. Lysates and supernatants of 293T cells transfected with either plasmid were harvested at 3 days post-transfection and subjected to immunoblotting using anti-preS1 and anti-preS2; anti-actin antibodies were used as a loading control. (C) Detection of serum antibodies binding to the preS1/2–47 peptide, preS2 peptide, HBs-L. HBs-M, and HBs-S proteins. Individual sera from mice (n=4) immunized with the indicated plasmids were diluted 100-fold for peptides and 250-fold for HBs proteins; the diluted sera then were added to ELISA microtiter wells containing each antigen. Bound antibody was detected using HRP-conjugated anti-mouse secondary antibody. The vertical axis for each serum and antigen shows absorbance in individual wells. (D and E) Neutralizing activities of sera from mice immunized with preS1 (D) or preS2 (E) expression plasmids. HBV/NL derived from genotype-C was preincubated with pooled sera (at 100-, 200-, 400-, 800-, and 1600-fold dilutions) derived from mice immunized with the indicated plasmids; the mixtures then were then used to infect G2/NT-18C cells for 16 h. Luciferase activity was determined at 7 days post-infection and is expressed relative to activity in reactions without serum. The statistical significance of differences between groups was evaluated using a Student's t-test (* P < 0.05, ** P < 0.01 vs. serum from empty vector-immunized control group). (F) HBVcc was mixed with indicated serum (100-fold dilutions) and used to inoculate HepG2-NTCPsec+ cells. HBsAg titers in the culture medium were measured. Statistical significance was evaluated using the Student's t-test (** P < 0.01 vs. no serum control). (G) HBV-positive cells were visualized by staining with an anti-HBc antibody, and nuclei were visualizedby staining with DAPI.

Article Snippet: The HBVcc-infected cells were fixed, permeabilized, and treated with rabbit polyclonal anti-HBc IgG (Austral Biologicals) followed by staining with Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Sequencing, Derivative Assay, Expressing, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Infection, Staining

Journal: Virus Research

Article Title: Identification of neutralizing epitopes in the preS2 domain of the hepatitis B virus

doi: 10.1016/j.virusres.2022.199014

Figure Lengend Snippet: Neutralization assay of anti-preS2 mAbs using HBV/NL derived from HBV genotype-C. (A) mAbs (0.5, 1, and 5 μg/mL) were incubated with HBV/NL (20 GEq/cell) for 1 h; the mixtures then were used to infect G2/NT-18C cells for 16 h. Luciferase activity of cells was determined at 7 days post-infection and is expressed relative to activity in cells infected with reactions performed without antibodies. Values represent the means of quadruplicate reactions; error bars indicate standard deviations. Statistical significance was evaluated using the Student's t-test (** P < 0.01 vs. no antibody control). (B) HBVcc was mixed with the indicated mAbs (1 μg/mL) and used to inoculate HepG2-NTCPsec+ cells. HBsAg titers in the culture medium were measured. Statistical significance was evaluated using the Student's t-test (** P < 0.01 vs. no antibody control). (C) HBV-positive cells were visualized by staining with an anti-HBc antibody; nuclei were visualized by staining with DAPI. (D) Epitope mapping of mAbs against preS2. Minimum epitopes recognized by each mAb are shown. Thirty-six synthetic overlapping 20-aa peptides corresponding to preS2 were used. The horizontal axis in each of the mAb panels shows absorbance units. The peptides showing antibody binding are indicated by an asterisk.

Article Snippet: The HBVcc-infected cells were fixed, permeabilized, and treated with rabbit polyclonal anti-HBc IgG (Austral Biologicals) followed by staining with Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Neutralization, Derivative Assay, Incubation, Luciferase, Activity Assay, Infection, Staining, Binding Assay