Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Hela, HepG2, and Huh7 cells were seeded in a 12-well plate for overnight, then left untreated or treated with human IFN-α (1,000 IU/ml) for 18 or 36 h. The untreated cells (control) were harvested together with the cells treated by IFN-α for 36 h. ISG20 expression was detected by Western blot. Hela cells transfected with plasmid F-ISG20 expressing the FLAG-tagged ISG20 were used as positive control for ISG20 Western blot (lane 2). β-actin expression was presented as loading control. (B) PHHs and HepG2 cells were cultured in 12-well-plate and treated with type I IFN-α (1,000 IU/ml), type II IFN-γ (100 ng/ml) or type III IFN-λ (100 ng/ml), or left untreated (control) for 2 days. The levels of ISG20 expression were determined by Western blot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Control, Expressing, Western Blot, Transfection, Plasmid Preparation, Positive Control, Cell Culture

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with either pHBV1.3 and F-ISG20 or empty vector, or pCMVHBV and F-ISG20 or empty vector, as indicated. Cells were harvested at day 5 post-transfection, and the levels of viral RNA and DNA were determined by Northern (top) and Southern (middle) blot hybridization, respectively. For RNA analysis, each lane was loaded with 10 μg of total RNA and probed with a genome-length, plus-strand-specific HBV riboprobe. Ribosomal RNAs (28S and 18S) are presented as loading controls. The positions of HBV pgRNA (3.5kb) and subgenomic surface RNAs (2.4kb and 2.1kb) are indicated. For DNA analysis, HBV core DNA was probed with genome-length, minus-strand-specific HBV riboprobe. The positions of relaxed circular (RC) and single-stranded (SS) DNAs are indicated. The relative pgRNA, sRNA or total DNA replicative intermediate level in each sample is expressed as the percentage of RNA or DNA of the cells transfected with empty vector. ISG20 overexpression was confirmed by Western blot using monoclonal antibodies against FLAG-tag. β-actin expression was presented as protein loading control (bottom panels). (B) The same experiment was done in Huh7 cells with pHBV1.3 as HBV expression vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Northern Blot, Hybridization, Over Expression, Western Blot, Bioprocessing, FLAG-tag, Expressing, Control

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepDES19 cells were seeded in 35 mm-dish and cultured with tet-free medium to induce HBV pgRNA transcription. 24 h later, cells were transfected with 4 μg of control vector or plasmid F-ISG20 for 36 h, then tet was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of FLAG-tagged ISG20 was detected by Western blot. The results are representative of three separate trials. (B) HepG2 cells in 12-well-plate were co-transfected with 0.7 μg of pTREHBVDES and 0.1 μg of pTet-off, plus 0.7 μg of control vector or plasmid F-ISG20. Four days post transfection, tet was added back and cells were harvested at indicated time points and subjected to HBV RNA qPCR analysis. The relative levels of HBV total RNA normalized to β-actin mRNA levels in each samples were expressed as the percentage of the RNA levels from the corresponding sample at 0 h time point (Mean ± SD, n = 4). The half-life of HBV RNA was marked on the plot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Cell Culture, Transfection, Control, Plasmid Preparation, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepDES19 cells were transfected with 100 nM of control siRNA (Lane 1–3) or ISG20 siRNA (siISG20) (lane 4–6) twice with a 24 h interval after tet being withdrawn. Culture medium was replaced 12 h after the 2nd siRNA transfection, and cells were either left untreated as controls (lane 1 &4) or treated with 100 IU/ml (lanes 2 & 5) or 1,000 IU/ml (lanes 3 & 6) of IFN-α. Cells were harvested 5 days after 2nd transfection. Viral total RNA (top panel), encapsidated pgRNA (upper middle panel), and core DNA (lower middle panel) were subjected to Northern and Southern analyses, respectively. ISG20 protein expression was revealed by Western blot, and β-actin served as loading control (bottom panels). The relative levels of viral nucleic acids and ISG20 expression in the siISG20 transfected or IFN-α treated samples (lanes 2–6) are expressed as the percentage of the control sample (lane 1). The data presented here are representative of two independent experiments.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2-NTCP12 cells stably transduced by control lentiviral shRNA (shcontrol) or ISG20 lentiviral shRNA (shISG20) were spinoculated with HBV at 100 vge/cell. 16 h later, the infected cells were mock treated or treated with 1,000 IU/ml of IFN-α for 6 days, and the cells were subjected to the following analyses: (A) The expression of ISG20 was analyzed by Western blot. (B) HBV infectivity was assessed by HBcAg immunofluorescence, and the percentage of HBcAg-positive cells were calculated from multiple microscopic field of view (mean±SD, n = 5). Nuclei were stained with DAPI. (C) HBV total RNA were quantified by qPCR and the relative expression levels to β-actin mRNA were plotted as fold change to control samples (HBV infected shcontrol cells without IFN-α treatment) (mean±SD, n = 3).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Stable Transfection, Control, shRNA, Infection, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were transfected with pHBV1.3 and equal amount of control vector (lanes 1 & 2) or F-ISG20 (lanes 3 & 4) or F-ISG20 D94G (lanes 5 & 6). Cells were harvested 5 days post-transfection and levels of HBV RNA (1st panel from the top) and encapsidated pgRNA (4th panel from the top) were determined by Northern blot hybridization. The assembled HBV capsid was revealed by native capsid gel EIA assay (3rd panel from the top) and the viral DNA in capsid was detected in situ by hybridization (5th panel from the top). HBV core DNA replicative intermediates were extracted and analyzed by Southern blot (6th panel from the top). Expression of FLAG-tagged ISG20 proteins was revealed by Western blot and β-actin served as loading control (bottom two panels). Results from duplicate experiments are presented.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Hybridization, Enzyme Immunoassay, In Situ, Southern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were co-transfected with plasmid pCMVHBVΔCΔP and either control vector (lane 1) or FLAG-Pol (lanes 2&3), or pCMVHBV with either control vector (lane 4) or F-ISG20 D94G (lanes 5&6). Cells were harvested 4 days post-transfection. Input HBV RNA was determined by Northern blot (top panels). Input FLAG-Pol and F-ISG20 D94G proteins were determined by Western blot using FLAG Ab (top panels). Cell lysates were immunoprecipitated with beads coated with FLAG Ab, the immunoprecipitated Pol and ISG20 D94G were revealed by Western blot using FLAG Ab (lanes 3&6, bottom panel), and the bound RNA was extracted by Trizol and analyzed by Northern blot (lanes 3&6, bottom panel). HA Ab pull-down served as negative controls (lanes 2 & 5, bottom panel). See for more experimental details.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Control, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells in 6-well-plate were co-transfected with 1 μg of pCMVHBVΔCΔP and 5 μg control vector (lane 1) or 1 μg of FLAG-Pol in the absence of HA-ISG20 D94G (lane 2) or increased amount of HA-ISG20 D94G (1 μg, 2 μg, 4 μg; lanes 3–5). The total amount of transfected DNA was kept constant (6 μg/well) by adding control vector plasmid (lanes 2–4). 5 days later, total cellular HBV RNA and protein (FLAG-Pol and HA-ISG20 D94G ) were determined by Northern and Western blot, respectively, as input controls (top panels). Immunoprecipitation was performed by using antibodies against HA or FLAG epitopes, followed by Northern blot analysis of HBV RNA (bottom panels).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of HBV pgRNA deletion clones. Plasmid pHBV1.3 contains a 1.3 overlength HBV genome (Genbank Accession Number U95551), starting at nt 1000. The HBV nucleotide positions are according to Galibert et al . Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5’ cap of mRNA; and the sawtooth line represents the polyA tail at the 3’ terminus of mRNA. The internal deletion clones (pg-IDs) are described in details in . The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 5’ or 3’ terminus of pgRNA coding sequences (pg-Δ5TR and pg-Δ3TR, respectively.), or both (pg-Δ5/3TR). The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector. (B) Sensitivity of HBV RNA with TR deletion to ISG20-mediated RNA reduction. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or F-ISG20 plasmid. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization. ( C) HBV TR insertion renders Luc gene to be sensitive to ISG20. The schematic illustration indicates the reporter construct EnII/Cp-Luc with HBV TR insertion at the flanking non-translational region of luciferase ORF. HepG2 cells were transfected with each indicated reporter plasmid and control vector or plasmid expressing ISG20. Cells were lysed at day 3 post transfection and luciferase activity was measured. The plotted relative luciferase activity (RLA) represents the mean ± SD (n = 3) of the percentage of absorbance obtained from wells transfected with ISG20 over control vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Control, Northern Blot, Hybridization, Construct, Luciferase, Expressing, Activity Assay

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic stem-loop structure of HBV ε RNA. Ribonucleotide sequences (nt 1847–1991, genotype D, subtype ayw) are presented with base paring indicated by dotted line. (B) Verification of the purified recombinant 6×His-tagged ISG20 by SDS-PAGE Coomassie staining. (C) EMSA assay of ISG20-ε binding. The indicated amount of ISG20 proteins were incubated with 100 ng 32 P-end-labeled ε RNA in binding buffer to form nucleoprotein complexes. Monoclonal anti-His antibody was used for supershifting of the His-ISG20/ HBV ε complex. Excessive amount of cold unlabeled HBV ε RNA (10×, 20×, 40×) were used to compete with the binding of ISG20 to 100 ng radiolabeled HBV ε. The nucleoprotein complexes were separated by native PAGE and the shifted bands were detected by autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Purification, Recombinant, SDS Page, Staining, Binding Assay, Incubation, Labeling, Clear Native PAGE, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustrations of the wildtype HBV ε RNA and mutants. The substructural domains of ε, including the lower stem, bulge, upper stem, and apical loop, are marked on the full-length form. Shorter versions of ε include the upper stem loop (US+L), lower stem with wildtype or mutant bulge sequence as loop (LS+B, LS+Bm), and LS+B with bottom 4 base-pairs removed from the lower stem (LSΔ4+B). These RNA fragments were chemically synthesized and 5’ end radiolabeled for ISG20 EMSA. (B) EMSA of ISG20 binding with full-length (FL) ε, US+L, and LS+B. (C) EMSA of ISG20 binding with LS+B, LS+Bm, and LS+B. (D) HepG2 cells were transfected with plasmid pMS transcribing the 2.1kb HBV RNA, or pMSΔ4bp transcribing the 2.1kb HBV with 4 nucleotides removed from the bottom right arm of the lower stem of ε, in the absence or presence of F-ISG20. HBV RNA and FLAG-tagged ISG20 were analyzed by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Mutagenesis, Sequencing, Synthesized, Binding Assay, Transfection, Plasmid Preparation, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of ISG20. The amino acid (a.a) positions are labeled with numbers. The gray boxes indicate the predicted Exo motifs. The enzymatic mutant site (D94G) is marked with an asterisk. (B) Bacterially expressed His-tagged ISG20 and mutants were purified and examined by SDS-PAGE Coomassie staining. The asterisk indicates a nonspecific protein band co-purified with the recombinant ΔExoII mutant. (C) EMSA of ε binding by wildtype ISG20 and the indicated mutants. (D) HepG2 cells were co-transfected with pHBV1.3 and control vector or indicated FLAG-ISG20 constructs. HBV RNA and ISG20 proteins were detected by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Labeling, Mutagenesis, Purification, SDS Page, Staining, Recombinant, Binding Assay, Transfection, Control, Plasmid Preparation, Construct, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: 0.1 µg of 5’-radiolabeled synthetic RNA substrates, specifically (A) the intact ε, upper stem-loop region (US+L), and lower stem with bulge serving as loop (LS+B); and (B) the intact ε, single-stranded left arm portion of ε, and 30-mer poly(rA), were incubated with the indicated amount of RNase A or purified His-ISG20 in nuclease reaction buffer for 15 min, then the reactions were terminated and the mixtures were fractionated through 10% TBE-Urea denaturing polyacrylamide gel, and the dried gel was subjected to autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Incubation, Purification, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: The major viral intermediates and products at each major HBV replication steps are illustrated. cccDNA (or other HBV transcription template)-derived 3.5kb RNA (including precore mRNA and pgRNA) and other shorter subgenomic RNA species (2.4/2.1kb surface mRNA and 0.7kb X mRNA) are aligned to show the location of ε on different RNA species. The black circle dots indicate the 5’ cap of mRNA, the zigzag lines represent the polyA tails. ISG20 is shown as a rectangle box and its targeting sites on HBV RNA are indicated by arrowheads. As a consequence of ISG20-mediated HBV RNA degradation, the illustrations and labels of viral proteins/antigens, pgRNA encapsidation and DNA replication are shown in gray color schemes. The solid gray triangles indicate capsid proteins, viral polymerase is shown in an oval shape before ε binding and then a gray circle dot in the nucleocapsids after pgRNA encapsidation.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Derivative Assay, Binding Assay

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Hela, HepG2, and Huh7 cells were seeded in a 12-well plate for overnight, then left untreated or treated with human IFN-α (1,000 IU/ml) for 18 or 36 h. The untreated cells (control) were harvested together with the cells treated by IFN-α for 36 h. ISG20 expression was detected by Western blot. Hela cells transfected with plasmid F-ISG20 expressing the FLAG-tagged ISG20 were used as positive control for ISG20 Western blot (lane 2). β-actin expression was presented as loading control. (B) PHHs and HepG2 cells were cultured in 12-well-plate and treated with type I IFN-α (1,000 IU/ml), type II IFN-γ (100 ng/ml) or type III IFN-λ (100 ng/ml), or left untreated (control) for 2 days. The levels of ISG20 expression were determined by Western blot.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Control, Expressing, Western Blot, Transfection, Plasmid Preparation, Positive Control, Cell Culture

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with either pHBV1.3 and F-ISG20 or empty vector, or pCMVHBV and F-ISG20 or empty vector, as indicated. Cells were harvested at day 5 post-transfection, and the levels of viral RNA and DNA were determined by Northern (top) and Southern (middle) blot hybridization, respectively. For RNA analysis, each lane was loaded with 10 μg of total RNA and probed with a genome-length, plus-strand-specific HBV riboprobe. Ribosomal RNAs (28S and 18S) are presented as loading controls. The positions of HBV pgRNA (3.5kb) and subgenomic surface RNAs (2.4kb and 2.1kb) are indicated. For DNA analysis, HBV core DNA was probed with genome-length, minus-strand-specific HBV riboprobe. The positions of relaxed circular (RC) and single-stranded (SS) DNAs are indicated. The relative pgRNA, sRNA or total DNA replicative intermediate level in each sample is expressed as the percentage of RNA or DNA of the cells transfected with empty vector. ISG20 overexpression was confirmed by Western blot using monoclonal antibodies against FLAG-tag. β-actin expression was presented as protein loading control (bottom panels). (B) The same experiment was done in Huh7 cells with pHBV1.3 as HBV expression vector.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Transfection, Plasmid Preparation, Northern Blot, Hybridization, Over Expression, Western Blot, Bioprocessing, FLAG-tag, Expressing, Control

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepDES19 cells were seeded in 35 mm-dish and cultured with tet-free medium to induce HBV pgRNA transcription. 24 h later, cells were transfected with 4 μg of control vector or plasmid F-ISG20 for 36 h, then tet was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of FLAG-tagged ISG20 was detected by Western blot. The results are representative of three separate trials. (B) HepG2 cells in 12-well-plate were co-transfected with 0.7 μg of pTREHBVDES and 0.1 μg of pTet-off, plus 0.7 μg of control vector or plasmid F-ISG20. Four days post transfection, tet was added back and cells were harvested at indicated time points and subjected to HBV RNA qPCR analysis. The relative levels of HBV total RNA normalized to β-actin mRNA levels in each samples were expressed as the percentage of the RNA levels from the corresponding sample at 0 h time point (Mean ± SD, n = 4). The half-life of HBV RNA was marked on the plot.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Cell Culture, Transfection, Control, Plasmid Preparation, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepDES19 cells were transfected with 100 nM of control siRNA (Lane 1–3) or ISG20 siRNA (siISG20) (lane 4–6) twice with a 24 h interval after tet being withdrawn. Culture medium was replaced 12 h after the 2nd siRNA transfection, and cells were either left untreated as controls (lane 1 &4) or treated with 100 IU/ml (lanes 2 & 5) or 1,000 IU/ml (lanes 3 & 6) of IFN-α. Cells were harvested 5 days after 2nd transfection. Viral total RNA (top panel), encapsidated pgRNA (upper middle panel), and core DNA (lower middle panel) were subjected to Northern and Southern analyses, respectively. ISG20 protein expression was revealed by Western blot, and β-actin served as loading control (bottom panels). The relative levels of viral nucleic acids and ISG20 expression in the siISG20 transfected or IFN-α treated samples (lanes 2–6) are expressed as the percentage of the control sample (lane 1). The data presented here are representative of two independent experiments.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Transfection, Control, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2-NTCP12 cells stably transduced by control lentiviral shRNA (shcontrol) or ISG20 lentiviral shRNA (shISG20) were spinoculated with HBV at 100 vge/cell. 16 h later, the infected cells were mock treated or treated with 1,000 IU/ml of IFN-α for 6 days, and the cells were subjected to the following analyses: (A) The expression of ISG20 was analyzed by Western blot. (B) HBV infectivity was assessed by HBcAg immunofluorescence, and the percentage of HBcAg-positive cells were calculated from multiple microscopic field of view (mean±SD, n = 5). Nuclei were stained with DAPI. (C) HBV total RNA were quantified by qPCR and the relative expression levels to β-actin mRNA were plotted as fold change to control samples (HBV infected shcontrol cells without IFN-α treatment) (mean±SD, n = 3).

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Stable Transfection, Control, shRNA, Infection, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were transfected with pHBV1.3 and equal amount of control vector (lanes 1 & 2) or F-ISG20 (lanes 3 & 4) or F-ISG20 D94G (lanes 5 & 6). Cells were harvested 5 days post-transfection and levels of HBV RNA (1st panel from the top) and encapsidated pgRNA (4th panel from the top) were determined by Northern blot hybridization. The assembled HBV capsid was revealed by native capsid gel EIA assay (3rd panel from the top) and the viral DNA in capsid was detected in situ by hybridization (5th panel from the top). HBV core DNA replicative intermediates were extracted and analyzed by Southern blot (6th panel from the top). Expression of FLAG-tagged ISG20 proteins was revealed by Western blot and β-actin served as loading control (bottom two panels). Results from duplicate experiments are presented.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Hybridization, Enzyme Immunoassay, In Situ, Southern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were co-transfected with plasmid pCMVHBVΔCΔP and either control vector (lane 1) or FLAG-Pol (lanes 2&3), or pCMVHBV with either control vector (lane 4) or F-ISG20 D94G (lanes 5&6). Cells were harvested 4 days post-transfection. Input HBV RNA was determined by Northern blot (top panels). Input FLAG-Pol and F-ISG20 D94G proteins were determined by Western blot using FLAG Ab (top panels). Cell lysates were immunoprecipitated with beads coated with FLAG Ab, the immunoprecipitated Pol and ISG20 D94G were revealed by Western blot using FLAG Ab (lanes 3&6, bottom panel), and the bound RNA was extracted by Trizol and analyzed by Northern blot (lanes 3&6, bottom panel). HA Ab pull-down served as negative controls (lanes 2 & 5, bottom panel). See for more experimental details.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Transfection, Plasmid Preparation, Control, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells in 6-well-plate were co-transfected with 1 μg of pCMVHBVΔCΔP and 5 μg control vector (lane 1) or 1 μg of FLAG-Pol in the absence of HA-ISG20 D94G (lane 2) or increased amount of HA-ISG20 D94G (1 μg, 2 μg, 4 μg; lanes 3–5). The total amount of transfected DNA was kept constant (6 μg/well) by adding control vector plasmid (lanes 2–4). 5 days later, total cellular HBV RNA and protein (FLAG-Pol and HA-ISG20 D94G ) were determined by Northern and Western blot, respectively, as input controls (top panels). Immunoprecipitation was performed by using antibodies against HA or FLAG epitopes, followed by Northern blot analysis of HBV RNA (bottom panels).

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of HBV pgRNA deletion clones. Plasmid pHBV1.3 contains a 1.3 overlength HBV genome (Genbank Accession Number U95551), starting at nt 1000. The HBV nucleotide positions are according to Galibert et al . Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5’ cap of mRNA; and the sawtooth line represents the polyA tail at the 3’ terminus of mRNA. The internal deletion clones (pg-IDs) are described in details in . The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 5’ or 3’ terminus of pgRNA coding sequences (pg-Δ5TR and pg-Δ3TR, respectively.), or both (pg-Δ5/3TR). The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector. (B) Sensitivity of HBV RNA with TR deletion to ISG20-mediated RNA reduction. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or F-ISG20 plasmid. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization. ( C) HBV TR insertion renders Luc gene to be sensitive to ISG20. The schematic illustration indicates the reporter construct EnII/Cp-Luc with HBV TR insertion at the flanking non-translational region of luciferase ORF. HepG2 cells were transfected with each indicated reporter plasmid and control vector or plasmid expressing ISG20. Cells were lysed at day 3 post transfection and luciferase activity was measured. The plotted relative luciferase activity (RLA) represents the mean ± SD (n = 3) of the percentage of absorbance obtained from wells transfected with ISG20 over control vector.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Control, Northern Blot, Hybridization, Construct, Luciferase, Expressing, Activity Assay

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic stem-loop structure of HBV ε RNA. Ribonucleotide sequences (nt 1847–1991, genotype D, subtype ayw) are presented with base paring indicated by dotted line. (B) Verification of the purified recombinant 6×His-tagged ISG20 by SDS-PAGE Coomassie staining. (C) EMSA assay of ISG20-ε binding. The indicated amount of ISG20 proteins were incubated with 100 ng 32 P-end-labeled ε RNA in binding buffer to form nucleoprotein complexes. Monoclonal anti-His antibody was used for supershifting of the His-ISG20/ HBV ε complex. Excessive amount of cold unlabeled HBV ε RNA (10×, 20×, 40×) were used to compete with the binding of ISG20 to 100 ng radiolabeled HBV ε. The nucleoprotein complexes were separated by native PAGE and the shifted bands were detected by autoradiography.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Purification, Recombinant, SDS Page, Staining, Binding Assay, Incubation, Labeling, Clear Native PAGE, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustrations of the wildtype HBV ε RNA and mutants. The substructural domains of ε, including the lower stem, bulge, upper stem, and apical loop, are marked on the full-length form. Shorter versions of ε include the upper stem loop (US+L), lower stem with wildtype or mutant bulge sequence as loop (LS+B, LS+Bm), and LS+B with bottom 4 base-pairs removed from the lower stem (LSΔ4+B). These RNA fragments were chemically synthesized and 5’ end radiolabeled for ISG20 EMSA. (B) EMSA of ISG20 binding with full-length (FL) ε, US+L, and LS+B. (C) EMSA of ISG20 binding with LS+B, LS+Bm, and LS+B. (D) HepG2 cells were transfected with plasmid pMS transcribing the 2.1kb HBV RNA, or pMSΔ4bp transcribing the 2.1kb HBV with 4 nucleotides removed from the bottom right arm of the lower stem of ε, in the absence or presence of F-ISG20. HBV RNA and FLAG-tagged ISG20 were analyzed by Northern and Western blot, respectively.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Mutagenesis, Sequencing, Synthesized, Binding Assay, Transfection, Plasmid Preparation, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of ISG20. The amino acid (a.a) positions are labeled with numbers. The gray boxes indicate the predicted Exo motifs. The enzymatic mutant site (D94G) is marked with an asterisk. (B) Bacterially expressed His-tagged ISG20 and mutants were purified and examined by SDS-PAGE Coomassie staining. The asterisk indicates a nonspecific protein band co-purified with the recombinant ΔExoII mutant. (C) EMSA of ε binding by wildtype ISG20 and the indicated mutants. (D) HepG2 cells were co-transfected with pHBV1.3 and control vector or indicated FLAG-ISG20 constructs. HBV RNA and ISG20 proteins were detected by Northern and Western blot, respectively.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Labeling, Mutagenesis, Purification, SDS Page, Staining, Recombinant, Binding Assay, Transfection, Control, Plasmid Preparation, Construct, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: 0.1 µg of 5’-radiolabeled synthetic RNA substrates, specifically (A) the intact ε, upper stem-loop region (US+L), and lower stem with bulge serving as loop (LS+B); and (B) the intact ε, single-stranded left arm portion of ε, and 30-mer poly(rA), were incubated with the indicated amount of RNase A or purified His-ISG20 in nuclease reaction buffer for 15 min, then the reactions were terminated and the mixtures were fractionated through 10% TBE-Urea denaturing polyacrylamide gel, and the dried gel was subjected to autoradiography.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Incubation, Purification, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: The major viral intermediates and products at each major HBV replication steps are illustrated. cccDNA (or other HBV transcription template)-derived 3.5kb RNA (including precore mRNA and pgRNA) and other shorter subgenomic RNA species (2.4/2.1kb surface mRNA and 0.7kb X mRNA) are aligned to show the location of ε on different RNA species. The black circle dots indicate the 5’ cap of mRNA, the zigzag lines represent the polyA tails. ISG20 is shown as a rectangle box and its targeting sites on HBV RNA are indicated by arrowheads. As a consequence of ISG20-mediated HBV RNA degradation, the illustrations and labels of viral proteins/antigens, pgRNA encapsidation and DNA replication are shown in gray color schemes. The solid gray triangles indicate capsid proteins, viral polymerase is shown in an oval shape before ε binding and then a gray circle dot in the nucleocapsids after pgRNA encapsidation.

Article Snippet: Endogenous ISG20 expression in HepG2-NTCP12 cells was stably knocked down by transduction with ISG20 shRNA lentiviral particles (Santa Cruz Biotechnology) per the manufacturer’s directions.

Techniques: Derivative Assay, Binding Assay