Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: A Y16F substitution in NS4A disrupts replication of an HCV subgenomic replicon in Huh7 cells, but not in Huh-7.5 cells. (A) Amino acid sequence of NS4A, with the Y16 residue starred and indicated in teal. Numbers correspond with the amino acid position within NS4A (amino acids 1 to 54) or the full-length HCV polyprotein (amino acids 1662 to 1715). Strain names are listed as found in the Los Alamos HCV sequence database. Conserved amino acids are indicated with a dot, whereas differences are listed. (B) Representative images of Huh-7.5 or Huh7 cells electroporated with the in vitro-transcribed HCV subgenomic replicon RNA (HP, genotype 1B; WT or Y16F). Cells were plated in serial dilutions (2 × 105, 2 × 104, and 2 × 103) and then stained with crystal violet after 3 weeks of G418 selection. Graphs show the relative transduction efficiency, which denote the percentages of colonies in Y16F-transduced cells relative to the WT. Bars indicate means ± the SEM (n = three to four biological replicates), with data analyzed by Student t test (*, P < 0.05; NS, not significant). (C) Immunoblot analysis of anti-NS4A immunoprecipitated extracts or whole-cell lysate (WCL) from 293T cells transfected with the indicated HCV proteins (genotype 1B) or vector (V). Panels are representative of three independent experiments.

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Sequencing, Residue, In Vitro, Staining, Selection, Transduction, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: RIG-I deletion in Huh7 cells does not restore HCV NS4A Y16F replication. Huh-7.5 cells (A) or Huh7 cells (B) were infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3). Immunoblot analysis was performed on lysates extracted at the indicated hours postinfection (hpi) or after mock infection (M). Graphs directly to the right of each blot (here and in panel F) show quantification of NS5A protein relative to tubulin at 72 hpi (mean ± the SEM; n = three biological replicates). (C) IFNB1 expression relative to HPRT1 from Huh7 cells infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 1) at 72 hpi as analyzed by RT-qPCR, with data displayed as means ± the SEM (n = three biological replicates). (D) Immunoblot of extracts of Huh7 and Huh7-RIG-I KO cells that were mock or SV infected (20 h). (E) IFN-β promoter reporter luciferase expression of Huh7 and Huh7-RIG-I KO cells expressing either vector or full-length RIG-I that were either mock or SV infected (20 h). Values show means ± the SD (n = three technical replicates) in RLU. (F) Huh7-RIG-I KO cells were infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3). Immunoblot analysis was performed on lysates extracted at the indicated times or mock infected (M). The graph directly to the right of this blot shows the quantification of NS5A protein relative to tubulin at 72 hpi (mean ± the SEM; n = 3 biological replicates). (G to I) Focus-forming assay of supernatants harvested from Huh-7.5 (G), Huh7 (H), and Huh7-RIG-I KO (clones 1 and 2) (I) cells at 72 hpi (MOI of 0.3). The data are presented as the percent HCV titer from the Y16F strain relative to the WT (set at 100%) and show means ± the SEM (n = two or three biological replicates). Data were analyzed by Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.005; NS, not significant).

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Focus Forming Assay, Clone Assay

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: HCV NS3-NS4A Y16F retains the ability to cleave MAVS. (A) Immunoblot analysis of lysates harvested from Huh7 cells expressing NS3-NS4A (WT, Y16F, or SA [NS3 active-site mutant S139A]) and vector (V) or Flag-MAVS. Arrows indicate the full-length (FL) and cleaved (C) forms of MAVS. (B) Immunoblot analysis of lysates harvested at 72 hpi from Huh-7.5, Huh7, or Huh7-MAVS KO cells that were either mock infected (M) or infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3). Arrows indicate the full-length (FL) and cleaved (C) forms of MAVS. (C) Intracellular HCV RNA levels in Huh7 cells expressing nontargeting shRNA (shNTV) or MAVS shRNA (shMAVS) infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3) and harvested at 72 hpi, as measured by RT-qPCR. The data are presented as relative HCV copies from Y16F relative to WT (set at 100%) and show the means ± the SEM (n = two biological replicates). Immunoblots to the right of the graph shows MAVS protein expression in the shNTV and shMAVS cell lines. Data were analyzed by Student t test (**, P < 0.01 or as indicated).

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Western Blot, Expressing, Mutagenesis, Plasmid Preparation, Infection, shRNA, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: IRF3 deletion in Huh7 cells restores HCV Y16F replication to the level of HCV WT. (A) Immunoblot of extracts of Huh7 and Huh7-IRF3 KO cells. (B) IFN-β promoter reporter luciferase expression of Huh7 and Huh7-IRF3 KO cells expressing either vector or IRF3 that were either mock or SV infected (20 h). Values show means ± the SD (n = three technical replicates) in RLU. (C) Immunoblot analysis of lysates harvested at 72 hpi from Huh7 and Huh7-IRF3 KO cells infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3). Graphs below each blot show quantification of NS5A protein relative to GAPDH (means ± the SEM; n = three biological replicates). (D) Focus-forming assay of supernatants harvested at 72 hpi from Huh7 or Huh7-IRF3 KO cells (clones 1 and 2) infected with HCV, WT, or NS4A Y16F (MOI of 0.3). The data are presented as the percent HCV titer from the Y16F strain relative to the WT (set at 100%) and show means ± the SEM (n = two biological replicates). Data were analyzed by Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.005; NS, not significant).

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Western Blot, Luciferase, Expressing, Plasmid Preparation, Infection, Focus Forming Assay, Clone Assay

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: Overexpression of Riplet reduces HCV NS4A Y16F replication in Huh-7.5 cells. (A) Immunoblot analysis of lysates harvested from Huh7 and Huh-7.5 cells for endogenous Riplet protein expression. The graph below the blot shows the means ± the SEM (n = three biological replicates) of quantification of Riplet protein expression relative to tubulin. (B) RNF135 (Riplet) expression relative to GAPDH from Huh7, Huh-7.5, and Huh-7.5+Riplet-V5 cells, as analyzed by RT-qPCR, with data displayed as means ± the SD (n = two to three technical replicates). Data were analyzed by one-way ANOVA analysis across the means of the three groups (***, P < 0.005). (C) Immunoblot analysis of lysates harvested from the indicated cell lines infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3) or mock infected (M) at 72 hpi. Two different exposures (light and dark) are shown for NS5A. Graphs below each blot show means ± the SEM (n = three biological replicates) of quantification of NS5A protein relative to tubulin. (D) Focus-forming assay of supernatants harvested at 72 hpi from the indicated cell lines infected with HCV, WT, or NS4A Y16F (MOI of 0.3). The data are presented as the percent HCV titer from the Y16F strain relative to the WT (set at 100%) and show means ± the SEM (n = three biological replicates). (E) RT-qPCR analysis of the indicated IFN-stimulated genes from RNA harvested from Huh-7.5+Riplet-V5 cells infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 1) at 48 hpi. The data are normalized to Huh-7.5+Riplet-V5 mock-infected cells, are presented as the induction of indicated genes (relative to HPRT1 with the WT level set at 1), and display means ± the SEM (n = two biological replicates). Data were analyzed by Student t test (*, P < 0.05; **, P < 0.05; ***, P < 0.005; NS, not significant).

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Over Expression, Western Blot, Expressing, Quantitative RT-PCR, Infection, Focus Forming Assay

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: HCV NS3-NS4A Y16F does not block IRF3 activation. (A) Confocal micrographs of Huh7 cells expressing either NS3-NS4A WT or Y16F (genotype 1B), or vector, that were either mock or SV infected (20 h) and immunostained with anti-IRF3 (green), anti-NS4A (red), or anti-SV (magenta). Nuclei were stained with Hoechst (blue). Scale bar, 10 μm. (B) Quantification of the percent cells positive for SV (and either WT or Y16F NS3-NS4A, as indicated) that had nuclear IRF3. The data are displayed as means ± the SEM (n = two biological replicates of >500 cells counted under each condition and replicate). Data were analyzed by one-way ANOVA (**, P < 0.01; ***, P < 0.005). (C) Immunoblot analysis of lysates from Huh7(–), Huh7-HP WT replicon, or Huh7-HP Y16F replicon cells, and a heat map (below) that shows the mean relative fold induction (SV infected/mock infected, relative to HPRT1) of specific genes, as measured by RT-qPCR analysis of RNA from mock- or SV-infected (20 h) Huh7, Huh7-HP WT replicon, or Huh7-HP Y16F replicon cells from three biological replicates.

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Blocking Assay, Activation Assay, Expressing, Plasmid Preparation, Infection, Staining, Western Blot, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: TBK1 inhibition restores HCV Y16F viral replication to the level of HCV WT. (A) Immunoblot analysis of lysates harvested from DMSO-treated or BX795 (1 or 10 μM)-treated Huh7 cells that were either mock-infected (M) or infected with SV (20 h). (B) Immunoblot analysis of lysates harvested at 72 hpi from Huh7 cells either pretreated for 1 h with DMSO or with BX795 (10 μM), infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3). Graphs below each blot show quantification of NS5A protein relative to GAPDH (means ± the SEM; n = three biological replicates). Data were analyzed by Student t test (*, P < 0.05; NS, not significant).

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Inhibition, Western Blot, Infection

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: Riplet interaction with HCV NS4A is reduced by the Y16F mutation. (A) Confocal micrographs of Huh7 cells expressing HA-Riplet and either NS3-NS4A WT or Y16F (genotype 1B), or vector, that were immunostained with anti-NS4A (green) and anti-HA (red), with the nuclei stained with Hoechst (blue). The zoom panel is taken from the images in the white boxes. Images are representative of ∼50 cells analyzed. Scale bar, 10 μm. (B) Immunoblot analysis of anti-HA (NS4A) immunoprecipitated extracts or whole-cell lysate (WCL) from Huh7 cells transfected with plasmids expressing Flag-Riplet and NS4A-HA (genotype 1B) WT or Y16F, or vector (–). The graph directly below shows means ± the SEM (n = three biological replicates) of the relative fold change of Flag-Riplet to NS4A-HA in the immunoprecipitated lanes. Data were analyzed by Student t test (*, P < 0.05).

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Staining, Western Blot, Immunoprecipitation, Transfection

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: The RING domain of Riplet regulates HCV Y16F virus replication. (A) Immunoblot analysis of Huh-7.5 cells expressing Flag-Riplet full-length (FL) or Flag-Riplet ΔRING. Two different exposures (light and dark) are shown for Flag. (B) Immunoblot analysis of lysates harvested at 72 hpi from Huh-7.5, Huh-7.5+Flag-Riplet FL, and Huh-7.5+Flag-Riplet ΔRING cells infected with HCV, WT, or NS4A Y16F (JFH1, MOI of 0.3). Graphs below the blot show quantification of NS5A protein relative to GAPDH (means ± the SEM; n = three biological replicates).

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Virus, Western Blot, Expressing, Infection

Journal: Journal of Virology

Article Title: Hepatitis C Virus Infection Is Inhibited by a Noncanonical Antiviral Signaling Pathway Targeted by NS3-NS4A

doi: 10.1128/JVI.00725-19

Figure Lengend Snippet: Oligonucleotides used for RT-qPCR and cloning

Article Snippet: Antibodies used for immunoblot and immunofluorescence analysis include: mouse anti-HCV NS4A (genotype 1B, 1:1,000; Virogen), mouse anti-HCV NS3 (genotype 1B, 1:1,000; Adipogen), mouse anti-HCV NS5A (genotype 2A, 1:1,000, clone 9e10; a gift from Charles Rice, Rockefeller University), mouse anti-Tubulin (1:5,000; Sigma), mouse anti-RIG-I (1:1,000; Adipogen), anti-Flag-HRP (1:2,500; Sigma), rabbit anti-Flag (1:1,000; Sigma), rabbit anti-MAVS (1:1,000; Bethyl Laboratories), mouse anti-IRF3 (1:1,000; a gift from Michael Gale, Jr., University of Washington [ 35 ]), rabbit anti-IRF3 (1:100; Cell Signaling Technology), rabbit anti-TBK1 (1:1,000; Cell Signaling Technology), rabbit anti-phospho-TBK1 (1:1,000; Cell Signaling Technology), mouse anti-V5 (1:1,000; Sigma), mouse anti-HA (1:1,000; Sigma), rabbit anti-GAPDH (1:1,000; Cell Signaling Technology), Hoescht (1:500; Thermo Fisher), Alexa Fluor-conjugated secondary antibodies (1:500, Life Technologies), rabbit anti-SV (1:1,000, MBL International), and mouse anti-Riplet (1:500; a gift from Sun Hur, Harvard University [ 20 ]).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Endogenous Hepatitis C Virus Homolog Fragments in European Rabbit and Hare Genomes Replicate in Cell Culture

doi: 10.1371/journal.pone.0049820

Figure Lengend Snippet: The cells infected with liver homogenates from domestic (DR), wild (WR) rabbits and a hare (H) were analyzed 14 days post infection. MDBK cells incubated with MAbs specific for NS3 (A to D), NS4A (E to H) and NS5 (I to L) proteins. BT cells incubated with MAbs specific for NS3 (M to P), NS4A (Q to T) and NS5 (U to X) proteins. Negative controls (NC) for each cell culture and each Mab was also performed (A, E, I, M, Q and U). No fluorescence was detected. Scale bars: (A to H), 20 µm; (I to X), 50 µm.

Article Snippet: The slides were then incubated with mouse MAbs anti-HCV NS3, anti-HCV NS4A and anti-HCV NS5 (all from Santa Cruz Biotechnology, Inc, Heidelberg, Germany) (1∶100) in blocking solution at room temperature for 60 min, and subsequently incubated with goat anti-mouse IgG-FITC (Santa Cruz Biotechnology, Inc, Heidelberg, Germany) (1∶200) for 30 min.

Techniques: Infection, Incubation, Cell Culture, Fluorescence

Journal: PLoS ONE

Article Title: Endogenous Hepatitis C Virus Homolog Fragments in European Rabbit and Hare Genomes Replicate in Cell Culture

doi: 10.1371/journal.pone.0049820

Figure Lengend Snippet: The cells infected with liver homogenates from domestic (DR), wild (WR) rabbits and a hare (H), were analyzed 14 days post infection. The cells were incubated with MAbs specific for NS3 (A to F), NS4A (H to M) and NS5 (O to T) proteins. High-magnification views of immunogold particles in several organelles are shown in boxes (C, E, G, J, L, N, Q, S and U). The immunogold particles were detected in vacuoles (v) (C and L), in mitochondria (mt) (G and J), cytoplasm (cy) (E, Q, S and U) and nucleous (n) (N). Arrows indicate the presence of gold particles detected in the same organelles (mt, cy) and close to the extracellular membrane (R). Negative controls (NC) for each MAb were also performed (A, H and O). No immunogold particles were detected. The immunogold particles were 10 nm in diameter. Scale bars: (A) 0.2 µm; (B, D, F, H, I, K, M, O, P, R and T), 200 nm; (C, E, G, J, L, N, Q, S and U), 100 nm.

Article Snippet: The slides were then incubated with mouse MAbs anti-HCV NS3, anti-HCV NS4A and anti-HCV NS5 (all from Santa Cruz Biotechnology, Inc, Heidelberg, Germany) (1∶100) in blocking solution at room temperature for 60 min, and subsequently incubated with goat anti-mouse IgG-FITC (Santa Cruz Biotechnology, Inc, Heidelberg, Germany) (1∶200) for 30 min.

Techniques: Infection, Incubation, Membrane

Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet: (A–D) 293T cell pools stably expressing Cas9 nuclease and an sgRNA targeting EMC6 were subsequently transiently co-transfected with constructs encoding HA-tagged DENV non-structural proteins and NS1-FLAG (A, C, and D) or GFP (B) as a transfection control. (E) ImageJ (NIH) was used to quantify band intensities for western blots of each of the DENV non-structural proteins in EMC6 knockout cells compared with wild-type from (A)–(D). Each dot represents a biological replicate. Bars represent mean ± SD. The Mann-Whitney U test was performed to assess statistical significance (*p < 0.05 and **p < 0.005 compared to NS1). (F–K) HEK293 cells were transfected with siRNAs against EMC1 or a scrambled negative control (scr). Forty-eight hours later, cells were transfected with a replication-defective full-length ZIKV cDNA (F and G) or with plasmids encoding individual S-tagged ZIKV non-structural proteins NS5 (H), NS4A (I), NS4B (J), or NS2B (K). The asterisk in (G) indicates a nonspecific background band. For (A)–(D) and (F)–(K) 24 h post-transfection, cells were lysed and proteins were separated using SDS-PAGE followed by western blotting for the indicated proteins. Each blot is representative of a minimum of two biological replicates. See also .

Article Snippet: Anti-DENV NS4A , Genetex , Cat# GTX124249; RRID:AB_11177084.

Techniques: Stable Transfection, Expressing, Transfection, Construct, Control, Western Blot, Knock-Out, MANN-WHITNEY, Negative Control, SDS Page

Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet: (A) The TMHMM version 2.0 algorithm was used to predict the presence of transmembrane helices in 2k-NS4B of DENV (UniProtKB: P29990). This plot depicts the probability of a given residue being located within a transmembrane helix, where greater values reflect greater transmembrane probability. The indicated amino acid positions on the x axis indicate the boundaries of each predicted transmembrane domain with scores above 0.3 for each predicted transmembrane domain. (B) HEK293T cells stably expressing Cas9 and sgRNA targeting EMC6 were co-transfected to express the indicated C-terminally GFP-tagged NS4B mutants and NS1-FLAG as a transfection control. 2k-96 indicates a C-terminal truncation at amino acid 97. 2k-58 indicates a C-terminal truncation at amino acid 59. Δ32–96 indicates a deletion from 32 to 96. Twenty-four hours post-transfection, cells were lysed and proteins were resolved using SDS-PAGE followed by western blotting for the indicated proteins. (C) Band intensitiesforNS4Bwild-type and Δ32–96 in EMC6 knockout cells compared with wild-type cells. Each point represents a biological replicate from cells transfected with NS4B-GFP (circles) or NS4B-HA (triangles). (D) Schematic of NS4B mutations to alter hydrophobicity of the pTM1 and pTM2 helices. Both of these helices are speculated to be membrane associated, with red representing hydrophobic and blue representing charged and polar hydrophilic residues, respectively (middle). Mutagenesis of the hydrophobic residues to lysine (top) is expected to result in decreased membrane association of pTM1 and pTM2. Conversely, mutagenesis of charged or polar residues to leucine (bottom) is expected to result in the insertion of pTM1 and pTM2 into the membrane. (E and F) Wild-type 293T cells or cells stably expressing Cas9 and sgRNA targeting EMC6 were transfected to express the indicated NS4B-HA mutants with substitutions to lysine residues (less hydrophobic) shown in (E) and substitutions to leucine residues (more hydrophobic) shown in (F). Twenty-four hours post-transfection, cells were lysed and proteins were resolved using SDS-PAGE followed by western blotting for the indicated proteins. Blots are representative of a minimum of three independent experiments.(G) Bands were quantitated using ImageJ and represented as a ratio of band intensity in EMC6 knockout versus wild-type cells. Each point represents a biological replicate, with bars representing mean ± SD. Triangles represent quantification from blots of NS4B with PNGase treatment, while circles represent quantification from blots of NS4B without PNGase treatment. Statistical significance was assessed using Dunnett’s test for multiple comparisons (**p < 0.005 and ***p < 0.0005 compared with WT NS4B). (H) Model of EMC interaction with theSec61 translocon and the flavivirus protein. Depicted is a flavivirus genomic RNA (upper left) being translated by a ribosome (blue) at the cytosolic face of the ER. Transmembrane domains are cotranslationally inserted into the ER membrane by the Sec61 translocon (brown). The EMC (yellow) is associated with the translocon and assists in the proper insertion and stabilization of certain multi-pass transmembrane domain proteins at the time of protein translation and/or translocation. The expression of both NS4A and NS4B of DENV and ZIKV is dependent on the EMC. Expression of the next protein in the flavivirus polypeptide, NS5, is also decreased in EMC-deficient cells when NS5 is expressed on the same polyprotein as NS4A and NS4B but not when expressed by itself.

Article Snippet: Anti-DENV NS4A , Genetex , Cat# GTX124249; RRID:AB_11177084.

Techniques: Residue, Stable Transfection, Expressing, Transfection, Control, SDS Page, Western Blot, Knock-Out, Membrane, Mutagenesis, Translocation Assay

Journal: Cell reports

Article Title: The ER Membrane Protein Complex Promotes Biogenesis of Dengue and Zika Virus Non-structural Multi-pass Transmembrane Proteins to Support Infection

doi: 10.1016/j.celrep.2019.04.051

Figure Lengend Snippet:

Article Snippet: Anti-DENV NS4A , Genetex , Cat# GTX124249; RRID:AB_11177084.

Techniques: Recombinant, Protease Inhibitor, Membrane, Luciferase, Transfection, Virus, Software

Journal: Biomolecules

Article Title: 1 H -Imidazole-2,5-Dicarboxamides as NS4A Peptidomimetics: Identification of a New Approach to Inhibit HCV-NS3 Protease

doi: 10.3390/biom10030479

Figure Lengend Snippet: The conservation of important hydrogen bonding in the structure-based design. ( A ) Interactions of NS4A (light green) with some NS3 residues (yellow). ( B ) Designed imidazole-2,5-dicarboxamide (light blue).

Article Snippet: The cofactor NS4A and the fluorescent fluorescein isothiocyanate NS4A (FITC-NS4A) were purchased from GenScript (Hong Kong, China).

Techniques:

Journal: Biomolecules

Article Title: 1 H -Imidazole-2,5-Dicarboxamides as NS4A Peptidomimetics: Identification of a New Approach to Inhibit HCV-NS3 Protease

doi: 10.3390/biom10030479

Figure Lengend Snippet: ( A ) Bar graph representation of changes in thermal stability (Δ T agg ) of NS3 when mixed with MOC derivatives or NS4A ( n = 3). ( B – E ) Plot graphs of protein aggregation (% light intensity) increase by increasing temperature (°C). The aggregation curve of NS3 domain alone is shown in the right bundles. The left bundles are for NS3 domain mixed with peptides as follows: ( B ) NS4A 21`–33` , ( C ) MOC-11, ( D ) MOC-23 and ( E ) MOC-24.

Article Snippet: The cofactor NS4A and the fluorescent fluorescein isothiocyanate NS4A (FITC-NS4A) were purchased from GenScript (Hong Kong, China).

Techniques:

Journal: Biomolecules

Article Title: 1 H -Imidazole-2,5-Dicarboxamides as NS4A Peptidomimetics: Identification of a New Approach to Inhibit HCV-NS3 Protease

doi: 10.3390/biom10030479

Figure Lengend Snippet: Inhibition of NS4A 21`–33` binding with NS3 by MOC compounds and NS3 inhibition. Standard error of mean (SEM), half maximal inhibitory concentration (IC 50 ).

Article Snippet: The cofactor NS4A and the fluorescent fluorescein isothiocyanate NS4A (FITC-NS4A) were purchased from GenScript (Hong Kong, China).

Techniques: Inhibition, Binding Assay, Concentration Assay

Journal: Biomolecules

Article Title: 1 H -Imidazole-2,5-Dicarboxamides as NS4A Peptidomimetics: Identification of a New Approach to Inhibit HCV-NS3 Protease

doi: 10.3390/biom10030479

Figure Lengend Snippet: Fluorescence anisotropy competition assay for MOC-24. The emitted fluorescence was measured (480/520 nm) proportional to the amount of bound FITC-NS4A 21`–33` (Y axis).

Article Snippet: The cofactor NS4A and the fluorescent fluorescein isothiocyanate NS4A (FITC-NS4A) were purchased from GenScript (Hong Kong, China).

Techniques: Fluorescence, Competitive Binding Assay

Journal: Biomolecules

Article Title: 1 H -Imidazole-2,5-Dicarboxamides as NS4A Peptidomimetics: Identification of a New Approach to Inhibit HCV-NS3 Protease

doi: 10.3390/biom10030479

Figure Lengend Snippet: The effect of MOC-24 on the NS3 activity ( n = 5). Neg_Ctrl = NS3 alone, MOC-24 = NS3 mixed with MOC-24 and Pos_Ctrl = NS3 mixed with NS4A 21`–33` . The emitted fluorescence (Y axis) are proportional to the catalytic activity of the enzyme ( n = 3).

Article Snippet: The cofactor NS4A and the fluorescent fluorescein isothiocyanate NS4A (FITC-NS4A) were purchased from GenScript (Hong Kong, China).

Techniques: Activity Assay, Fluorescence