Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 1. HCV NS5A induces the engulfment of mitochondria within autophagic vacuoles: (A) Huh7 cells were transduced with lentiviruses expressing RFP-LC3 and Mito-GFP, as described in

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Expressing

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 2. HCV NS5A induces the translocation of Parkin to mitochondria: (A) Huh7 cells were trans- duced with lentiviruses harboring RFP-Parkin and Mito-GFP, according to the procedure described

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Translocation Assay

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 3. HCV NS5A induces mitolysosome formation: (A) Huh7 cells were transduced with pTRIP- Mito-QC lentiviruses, as described in the “Materials and Methods” section, generating Huh7/Mito- QC cells. Huh7/Mito-QC cells were transduced with (+) or without (−) pTRIP-HCV NS5A-miRFP670 lentiviruses. After forty-eight hours, the cells were analyzed via confocal microscopy. (B) The number of RFP+/GFP−mitolysosomes was quantified with Image J, as described previously [33]. The data are presented as means ± SEMs (n = 10, *** p < 0.001). (C) The selected live imaging frames show the magnified area in the white dashed box of the top panel in (A). The white arrowheads indicate the formation of RFP+/GFP−mitolysosomes. (D–F) CLEM analysis of mitolysosome formation in HCV NS5A-expressing cells. (D) Huh7/Mito-QC cells were transduced with lentiviruses expressing HCV NS5A-miRFP670 for forty-eight hours and then processed for confocal microscopy. (E) The Z-stacks of the confocal micrograph shown in (D) were assembled and deconvoluted into a 3-D image. The white dashed boxes indicate the loci of RFP+/GFP−mitolysosomes. (F) The aligned IF and CLEM image of the cells from (D) is shown. The white dashed boxes in the left panel are enlarged and shown in the magnified images in the right panel. The white arrowheads indicate the loci of mitolysosomes.

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Confocal Microscopy, Imaging, Expressing

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 4. HCV NS5A enhances mitophagic flux and induces ubiquitin recruitment to mitochondria: (A) Huh7 cells were transduced with pTRIP-MT-Keima lentiviruses, according to the procedure

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Ubiquitin Proteomics, Transduction

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 5. HCV NS5A induces ubiquitin Ser65 phosphorylation and PINK1 stabilization in mitochon- dria: (A,B) (A) Huh7/RFP-LC3/Mito-GFP cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses. Forty-eight hours later, the cells were immunostained with a phospho- ubiquitin (Ub; Ser65) antibody and analyzed via confocal microscopy. (B) Foci of phospho-ubiquitin (Ser65) recruited onto mitophagosomes in which RFP-LC3 puncta sequestered Mito-GFP-labeled

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Transduction, Confocal Microscopy, Labeling

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 6. Recruitment of NDP52 and OPTN into HCV NS5A-activated mitophagy: (A,B) (A) Huh7 cells were transduced with lentiviruses expressing RFP-LC3, Mito-miRFP670, or GFP-NDP52, as described in the “Materials and Methods” section, generating Huh7/RFP-LC3/Mito-miRFP670/GFP- NDP52 cells. Then, the cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (B) The number of GFP-NDP52

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Expressing, Confocal Microscopy

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 7. Recruitment of ATG5 into close proximity to mitochondria for HCV NS5A-induced mi- tophagy: (A) Huh7 cells were transduced with lentiviruses expressing RFP-LC3 and mito-miRFP670

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Expressing

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 8. Recruitment of DFCP1 into the proximity of mitochondria for HCV NS5A-induced mi- tophagy: (A) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-DFCP1, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-DFCP1 cells. Then, Huh7/RFP- LC3/Mito-miRFP670/GFP-DFCP1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A- mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (B) The number of GFP-DFCP1 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. (C) The frames of selected live images show the magnified area in the white dashed box of the top panel in (A). The white arrowheads indicate the recruitment of GFP-DFCP1 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. (D) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-DFCP1, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-DFCP1 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-DFCP1 cells were transduced with (+) or without (−) pTRIP-

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Expressing, Confocal Microscopy, Labeling

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 9. Translocation of ATG14 into close proximity to mitochondria for HCV NS5A-induced mitophagy: (A) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG14, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG14 cells. Then, Huh7/RFP- LC3/Mito-miRFP670/GFP-ATG14 cells were transduced with (+) or without (−) pTRIP-HCV NS5A- mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (B) The number of GFP-ATG14 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequester Mito- miRFP670-expressing mitochondria, was quantified. (C) The selected live imaging frames show the magnified area in the white dashed box of the top panel in (A). The white arrowheads indicate the recruitment of GFP-ATG14 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. (D) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG14, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG14 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG14 cells were transduced with (+) or without (−)

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Translocation Assay, Transduction, Expressing, Confocal Microscopy, Imaging, Labeling

Journal: Pathogens (Basel, Switzerland)

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation.

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Figure 10. Translocation of ULK1 into the proximity of mitochondria for HCV NS5A-activated mitophagy: (A) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses express- ing GFP-ULK1, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ULK1 cells. Then, Huh7/RFP- LC3/Mito-miRFP670/GFP-ULK1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A- mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (B) The number of GFP-ULK1 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. (C) The selected live imaging frames show the magnified area in the white dashed box of the top panel in (A). The white arrowheads indicate the recruitment of GFP-ULK1 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. (D) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ULK1, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ULK1 cells. Then, Huh7/RFP- Parkin/Mito-miRFP670/GFP-ULK1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (E) The number of GFP-ULK1 molecules recruited to the RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. (F) The selected live imaging frames show the magnified area in the white dashed box of the top panel in (D). The white arrowheads indicate the recruitment of GFP-ULK1 to Mito-miRFP670-labeled mitochondria after RFP-Parkin translocation. The data shown in (B,E) represent the mean ± SEM (n = 10, *** p < 0.001).

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [34]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Translocation Assay, Transduction, Confocal Microscopy, Expressing, Imaging, Labeling

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: HCV NS5A induces the engulfment of mitochondria within autophagic vacuoles: ( A ) Huh7 cells were transduced with lentiviruses expressing RFP-LC3 and Mito-GFP, as described in the “Materials and Methods” section, to generate Huh7/RFP-LC3/Mito-GFP cells. Then, Huh7/RFP-LC3/Mito-GFP cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The degree of colocalization between RFP-LC3-labeled autophagic vacuoles and Mito-GFP-expressing mitochondria was quantified. The data are presented as means ± SEMs ( n = 10, *** p < 0.001). ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the sequestration of Mito-GFP-labeled mitochondria by RFP-LC3 puncta. ( D – G ) CLEM analysis of mitochondrial sequestration by autophagic vacuoles in HCV NS5A-expressing cells. ( D ) Huh7/RFP-LC3/Mito-GFP cells were transduced with lentiviruses expressing HCV NS5A-mTagBFP2 for forty-eight hours and then processed for confocal microscopy. ( E ) The assembled Z-stacks of the confocal micrographs in ( D ) were reconstituted into a 3-D image. The white dashed boxes indicate the engulfment of Mito-GFP-expressing mitochondria within RFP-LC3 puncta. ( F ) The aligned image of the confocal micrograph (IF) and electron micrograph (EM) from the CLEM analysis of cells in ( D ) is shown. The white dashed boxes in the left panel are enlarged and shown in the magnified images in the right panel. The white arrowheads indicate the sequestration of mitochondria within autophagic vacuoles. ( G ) The enlarged images show the magnified white dashed boxes in the EM of ( F ). The white arrows indicate the phagophores wrapped around deformed mitochondria.

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Expressing, Confocal Microscopy, Labeling, Imaging

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: HCV NS5A induces the translocation of Parkin to mitochondria: ( A ) Huh7 cells were transduced with lentiviruses harboring RFP-Parkin and Mito-GFP, according to the procedure described in the “Materials and Methods” section, to establish Huh7/RFP-Parkin/Mito-GFP cells. Huh7/RFP-LC3/Mito-GFP cells were transduced with (+) or without (−) pTRIP-HCV NS5A-miRFP670 lentiviruses for forty-eight hours and then analyzed via confocal microscopy. ( B ) The degree of colocalization between RFP-LC3-Parkin and Mito-GFP-labeled mitochondria was quantified. The data are presented as means ± SEMs ( n = 10, *** p < 0.001). ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the translocation of RFP-Parkin to Mito-GFP-expressing mitochondria. ( D – F ) CLEM analysis of the mitochondrial translocation of RFP-Parkin in HCV NS5A-expressing cells. ( D ) Huh7/RFP-Parkin/Mito-GFP cells were transduced with lentiviruses expressing HCV NS5A-miRFP670. Forty-eight hours later, the cells were analyzed via confocal microscopy. ( E ) The Z-stacks of the confocal micrograph shown in ( D ) were assembled and deconvoluted into a 3-D image. The white dashed boxes indicate the Mito-GFP-expressing mitochondria with RFP-Parkin translocation. ( F ) The aligned IF and CLEM image of the cells from ( D ) is presented. The white dashed boxes in the left panel are enlarged and shown in the magnified images in the right panel. The white arrowheads indicate the degradative mitochondria in which Parkin translocates.

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Translocation Assay, Transduction, Confocal Microscopy, Labeling, Imaging, Expressing

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: HCV NS5A induces mitolysosome formation: ( A ) Huh7 cells were transduced with pTRIP-Mito-QC lentiviruses, as described in the “Materials and Methods” section, generating Huh7/Mito-QC cells. Huh7/Mito-QC cells were transduced with (+) or without (−) pTRIP-HCV NS5A-miRFP670 lentiviruses. After forty-eight hours, the cells were analyzed via confocal microscopy. ( B ) The number of RFP + /GFP − mitolysosomes was quantified with Image J, as described previously . The data are presented as means ± SEMs ( n = 10, *** p < 0.001). ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the formation of RFP + /GFP − mitolysosomes. ( D – F ) CLEM analysis of mitolysosome formation in HCV NS5A-expressing cells. ( D ) Huh7/Mito-QC cells were transduced with lentiviruses expressing HCV NS5A-miRFP670 for forty-eight hours and then processed for confocal microscopy. ( E ) The Z-stacks of the confocal micrograph shown in ( D ) were assembled and deconvoluted into a 3-D image. The white dashed boxes indicate the loci of RFP + /GFP − mitolysosomes. ( F ) The aligned IF and CLEM image of the cells from ( D ) is shown. The white dashed boxes in the left panel are enlarged and shown in the magnified images in the right panel. The white arrowheads indicate the loci of mitolysosomes.

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Confocal Microscopy, Imaging, Expressing

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: HCV NS5A enhances mitophagic flux and induces ubiquitin recruitment to mitochondria: ( A ) Huh7 cells were transduced with pTRIP-MT-Keima lentiviruses, according to the procedure described in the “Materials and Methods” section, to establish Huh7/MT-Keima cells. Then, Huh7/MT-Keima cells were transduced with (+) or without (−) pTRIP-HCV NS5A-miRFP670 lentiviruses. Forty-eight hours later, the cells were analyzed via confocal microscopy at short (488 nm) and long (561 nm) excitation wavelengths. ( B ) The percentage of cells containing acidic MT-Keima (excitation at 561 nm) was quantified, as described previously . The data are presented as means ± SEMs ( n = 10, *** p < 0.001). ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the cells expressing acidic MT-Keima. ( D ) Huh7 cells were transduced with lentiviruses expressing RFP-LC3, GFP-Ub, or Mito-miRFP670, as described in the “Materials and Methods” section, generating Huh7/RFP-LC3/GFP-Ub/Mito-miRFP670 cells. Huh7/RFP-LC3/GFP-Ub/Mito-miRFP670 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and then analyzed via confocal microscopy. ( E ) The number of RFP-LC3 puncta containing GFP-Ub on Mito-miRFP670-labeled mitochondria was quantified. The data are presented as means ± SEMs ( n = 10, *** p < 0.001). ( F ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-Ub to Mito-miRFP670-expressing mitochondria and the subsequent sequestration of RFP-LC3-labeled autophagic vacuoles.

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Ubiquitin Proteomics, Transduction, Confocal Microscopy, Imaging, Expressing, Labeling

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: HCV NS5A induces ubiquitin Ser65 phosphorylation and PINK1 stabilization in mitochondria: ( A , B ) ( A ) Huh7/RFP-LC3/Mito-GFP cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses. Forty-eight hours later, the cells were immunostained with a phospho-ubiquitin (Ub; Ser65) antibody and analyzed via confocal microscopy. ( B ) Foci of phospho-ubiquitin (Ser65) recruited onto mitophagosomes in which RFP-LC3 puncta sequestered Mito-GFP-labeled mitochondria were quantified. ( C , D ) ( C ) Huh7/RFP-Parkin/Mito-GFP cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours. Then, the cells were immunostained with an anti-phospho-ubiquitin (Ser65) antibody and analyzed via confocal microscopy. ( D ) Foci of phosphor-Ub recruited to RFP-Parkin-translocated Mito-GFP-expressing mitochondria were quantified. ( E , F ) ( E ) Huh7/RFP-LC3/Mito-GFP cells were transduced with lentiviruses expressing PINK1-miRFP670, generating Huh7/RFP-LC3/Mito-miRFP670/PINK1-miRFP670 cells. Then, the cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( F ) Foci of PINK1-miRFP670 stabilized on mitophagosomes, in which RFP-LC3 puncta sequestered Mito-GFP-expressing mitochondria were quantified. ( G , H ) ( G ) Huh7/RFP-Parkin/Mito-GFP cells were transduced with lentiviruses expressing PINK1-miRFP670 to establish Huh7/RFP-LC3/Mito-miRFP670/PINK1-miRFP670 cells. Then, the cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( H ) The number of MiRFP670-PINK1 foci recruited onto the RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. The data shown in ( B , D , F , H ) represent the mean ± SEM ( n = 10, *** p < 0.001). The magnified field-1 and magnified field-2 in ( A , C , E , G ) show enlarged images of white dashed boxes 1 and 2 in the top and bottom panels. The white arrowheads indicate colocalized signals.

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Transduction, Confocal Microscopy, Labeling, Expressing

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Recruitment of NDP52 and OPTN into HCV NS5A-activated mitophagy: ( A,B ) ( A ) Huh7 cells were transduced with lentiviruses expressing RFP-LC3, Mito-miRFP670, or GFP-NDP52, as described in the “Materials and Methods” section, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-NDP52 cells. Then, the cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-NDP52 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. ( C , D ) ( C ) Huh7 cells were transduced with lentiviruses expressing RFP-LC3, Mito-miRFP670, or GFP-OPTN, as described in the “Materials and Methods” section, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-OPTN cells. Then, the cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( D ) The number of GFP-OPTN molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. ( E , F ) ( E ) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with lentiviruses expressing GFP-NDP52, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-NDP52 cells. Then, the cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( F ) The number of GFP-NDP52 molecules recruited to RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( G , H ) ( G ) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with lentiviruses expressing GFP-OPTN to establish Huh7/RFP-Parkin/Mito-miRFP670/GFP-OPTN cells. Then, the cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. (H) The number of GFP-OPTN molecules recruited to RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. The data shown in ( B , D , F , H ) represent the mean ± SEM ( n = 10, *** p < 0.001). The magnified field-1 and magnified field-2 in ( A , C , E , G ) show enlarged images of white dashed boxes 1 and 2 in the top and bottom panels. The white arrowheads indicate colocalized signals.

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Expressing, Confocal Microscopy, Labeling

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Recruitment of ATG5 into close proximity to mitochondria for HCV NS5A-induced mitophagy: ( A ) Huh7 cells were transduced with lentiviruses expressing RFP-LC3 and mito-miRFP670 to establish Huh7/RFP-LC3/Mito-miRFP670 cells. Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG5, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG5 cells. Then, Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG5 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-ATG5 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-ATG5 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. ( D ) Huh7 cells were transduced with lentiviruses expressing RFP-Parkin and Mito-miRFP670 to establish Huh7/RFP-Parkin/Mito-miRFP670 cells. Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG5, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG5 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG5 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( E ) The number of GFP-ATG5 molecules recruited to RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( F ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( D ). The white arrowheads indicate the recruitment of GFP-ATG5 to Mito-miRFP670-labeled mitochondria after translocation by RFP-Parkin. The data shown in ( B , E ) represent the mean ± SEM ( n = 10, *** p < 0.001).

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Expressing, Confocal Microscopy, Imaging, Labeling, Translocation Assay

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Recruitment of DFCP1 into the proximity of mitochondria for HCV NS5A-induced mitophagy: ( A ) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-DFCP1, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-DFCP1 cells. Then, Huh7/RFP-LC3/Mito-miRFP670/GFP-DFCP1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-DFCP1 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. ( C ) The frames of selected live images show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-DFCP1 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. ( D ) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-DFCP1, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-DFCP1 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-DFCP1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( E ) The number of GFP-DFCP1 molecules recruited to RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( F ) The frames of selected live images show the magnified area in the white dashed box of the top panel in ( D ). The white arrowheads indicate the recruitment of GFP-DFCP1 to Mito-miRFP670-labeled mitochondria after translocation by RFP-Parkin. The data shown in ( B , E ) represent the mean ± SEM ( n = 10, *** p < 0.001).

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Transduction, Expressing, Confocal Microscopy, Labeling, Translocation Assay

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Translocation of ATG14 into close proximity to mitochondria for HCV NS5A-induced mitophagy: ( A ) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG14, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG14 cells. Then, Huh7/RFP-LC3/Mito-miRFP670/GFP-ATG14 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-ATG14 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequester Mito-miRFP670-expressing mitochondria, was quantified. ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-ATG14 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. ( D ) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ATG14, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG14 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-ATG14 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( E ) The number of GFP-ATG14 molecules recruited to RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( F ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( D ). The white arrowheads indicate the recruitment of GFP-ATG14 to Mito-miRFP670-labeled mitochondria after RFP-Parkin translocation. The data shown in ( B , E ) represent the mean ± SEM ( n = 10, *** p < 0.001).

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Translocation Assay, Transduction, Expressing, Confocal Microscopy, Imaging, Labeling

Journal: Pathogens

Article Title: Hepatitis C Virus NS5A Activates Mitophagy Through Cargo Receptor and Phagophore Formation

doi: 10.3390/pathogens13121139

Figure Lengend Snippet: Translocation of ULK1 into the proximity of mitochondria for HCV NS5A-activated mitophagy: ( A ) Huh7/RFP-LC3/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ULK1, generating Huh7/RFP-LC3/Mito-miRFP670/GFP-ULK1 cells. Then, Huh7/RFP-LC3/Mito-miRFP670/GFP-ULK1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( B ) The number of GFP-ULK1 molecules recruited onto mitophagosomes, in which RFP-LC3 puncta sequestered Mito-miRFP670-expressing mitochondria, was quantified. ( C ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( A ). The white arrowheads indicate the recruitment of GFP-ULK1 to Mito-miRFP670-labeled mitochondria before sequestration by RFP-LC3 puncta. ( D ) Huh7/RFP-Parkin/Mito-miRFP670 cells were transduced with retroviruses expressing GFP-ULK1, generating Huh7/RFP-Parkin/Mito-miRFP670/GFP-ULK1 cells. Then, Huh7/RFP-Parkin/Mito-miRFP670/GFP-ULK1 cells were transduced with (+) or without (−) pTRIP-HCV NS5A-mTagBFP2 lentiviruses for forty-eight hours and analyzed via confocal microscopy. ( E ) The number of GFP-ULK1 molecules recruited to the RFP-Parkin-translocated Mito-miRFP670-labeled mitochondria was quantified. ( F ) The selected live imaging frames show the magnified area in the white dashed box of the top panel in ( D ). The white arrowheads indicate the recruitment of GFP-ULK1 to Mito-miRFP670-labeled mitochondria after RFP-Parkin translocation. The data shown in ( B , E ) represent the mean ± SEM ( n = 10, *** p < 0.001).

Article Snippet: To construct pmTagBFP2-N1-HCV NS5A and pmiRFP670-N1-HCV NS5A, a polymerase chain reaction (PCR)-amplified HCV NS5A gene fragment from the HCV JFH1 infectious clone (genotype 2a, kindly provided by Takaji Wakita [National Institute of Infectious Disease, Tokyo, Japan] [ ]) was subcloned and inserted into pmTagBFP2-N1 (#54566, Addgene) and pmiRFP670-N1 (#79987, Addgene).

Techniques: Translocation Assay, Transduction, Expressing, Confocal Microscopy, Imaging, Labeling

Journal: Cell reports

Article Title: Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress.

doi: 10.1016/j.celrep.2024.114069

Figure Lengend Snippet: Figure 5. Stress-mediated activation of the p38-MAPK/MK2 and PI3K/Akt signaling pathways mediates TTP and Brf1 phosphorylation (A and B) Analysis of p38-MAPK/MK2 and PI3K/Akt kinase signaling pathways upon HCV infection and IFN-a treatment (n = 3). (A) Representative western blot. (B) Corresponding quantification of p-eIF2a, p-p38, p-Akt, GADD34, and HCV NS5A levels, all normalized to eIF3b and represented as arbitrary units (a.u.) (mean ± SD). Statistical significance compared with mock was calculated using upaired, two-sided Student’s t-test (*p < 0.05, ***p < 0.001, ****p < 0.0001). (C and D) Dynamics of p38-MAPK/MK2 and PI3K/Akt pathway activation upon thapsigargin treatment for up to 6 h (n = 3). (C) Representative western blot. (D) Corresponding quantifications (mean ± SD). Statistical significance compared with DMSO is indicated. (E and F) Analysis of TTP and Brf1 phosphorylation levels by Phos-tag gel analysis (n = 3). Cells transiently expressing myc-his-tagged TTP (E) or Brf1 (F) were treated with DMSO or thapsigargin for the indicated times. lPPase, l phosphatase; HP, hyperphosphorylated form; NP, non-phosphorylated form. Shown are representative western blot analyses (left) and corresponding quantifications (right). Level of hyperphosphorylated forms is represented as a percentage of the total signal intensity in the corresponding lane (mean ± SD). Statistical significance compared with DMSO (0) is indicated. See also Figure S5. Statistical significance for (D)–(F) was calculated using one-way ANOVA with Dunnett’s multiple comparisons (n.s., non-significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: The following primary antibodies were used at the indicated dilutions and incubated overnight at 4 C : anti-GADD34 (Proteintech, Cat# 10449-I-AP; 1:1,000; milk), anti-eIF2a (Cell Signaling, Cat# 9722; 1:1,000; milk), anti-phospho-eIF2a (Cell Signaling, Cat# 3398; 1:1,000; BSA), anti-p38 (Cell Signaling, Cat# 8690; 1:1,000 milk), anti-phospho-p38 (Cell Signaling, 4511, 1:1,000; BSA), anti-Akt (Cell Signaling, Cat# 4691; 1:1,000; milk), anti-phospho-Akt (R&D systems, Cat# AF887; 1:1,000; BSA), anti-HCV NS5A (NS5A 9E10 was a gift from Charles Rice; 1:2,000; milk), anti-GAPDH (Santa Cruz, Cat# sc-365062; 1:5,000; milk), anti-eIF3a (Bethyl Laboratories, Cat# A302-002A; 1:5,000; milk), anti-eIF3b (Bethyl Laboratories, Cat# A301-761A; 1:5,000; milk), anti-myc-tag (Cell Signaling, Cat# 2276; 1:1,000; milk), anti-puromycin 12D10 (Millipore, Cat# MABE343; 1:2000; milk), antiTristetraprolin (TTP) D1I3T (Cell Signaling, Cat# 71632; 1:500; milk), anti-ZFP36L1 (Brf1) (Thermo Fisher Scientific, Cat# PA568973; 1:500; milk).

Techniques: Activation Assay, Protein-Protein interactions, Phospho-proteomics, Infection, Western Blot, Expressing

Journal: Kobe Journal of Medical Sciences

Article Title: Transcription Factor JunB Suppresses Hepatitis C Virus Replication

doi:

Figure Lengend Snippet: (A) Huh-7.5 cells were infected with HCV J6/JFH1 at a multiplicity of infection (MOI) of 1. At 2, 4, and 6 days postinfection (dpi), the cells were harvested. The cell lysates were analyzed by immunoblotting with the indicated antibodies. The level of GAPDH served as a loading control. (B) Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 1. At 6 dpi, the cells were harvested with or without pretreatment with either JNK inhibitor SP600125 (30 μM for 30 h) or antioxidant N-acetyl cysteine (NAC; 5 mM for 8 h). The cell lysates were analyzed by immunoblotting with the indicated antibodies. The level of GAPDH served as a loading control. (C) Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 1 with and without treatment of HCV NS5A inhibitor (daclatasvir; 1 μM). At 2 and 4 dpi, the cells were harvested, and the cell lysates were analyzed by immunoblotting with the indicated antibodies. The level of GAPDH served as a loading control.

Article Snippet: HCV NS5A inhibitor daclatasvir (ChemScene, Monmouth Junction, NJ), antioxidant N -acetyl cystein (NAC) (Sigma, St. Louis, MO), and JNK inhibitor SP600125 (Selleck Chemicals, Houston, TX, USA) were used.

Techniques: Infection, Western Blot, Control