Journal: Journal of Virology

Article Title: The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle

doi: 10.1128/jvi.00180-23

Figure Lengend Snippet: Role of ERES and ERGIC in HCV entry and replication. (A) Bar graph depicting the relative fold change in luciferase reporter activity in SEC16A, ERGIC-53, TFG, and Claudin-1 (CLDN-1) knockdown cells transduced with HCV pseudotyped particles (HCVpp). (B) Western blot analysis depicting the expression status of cell surface receptors in Huh-7 cells knockdown for SEC16A, ERGIC-53, TFG, and Claudin-1. GAPDH was used as an internal loading control. (C) Bar graphs representing the relative fold change in luciferase reporter activity in HCV genotype 2a subgenomic replicon (SGR-Feo) cells transfected with siNT, siSEC16A, siERGIC-53, and siTFG at 48 h posttransfection. (D) Western blot analysis depicting the knockdown efficiency from the subgenomic replicon cells transfected with respective siRNA. (E) Illustration representing the experimental timeline and approach used to determine the role of ERES, ERGIC, and TFG in HCV replication in the HCV cell culture model. (F) Bar graphs representing the fold change relative to siNT in HCV genome copies at 18 hpi. (G) Immunofluorescence images and Western blot analysis depicting the overexpression of SEC16A-GFP or EV-GFP and the SEC16A knockdown efficiency. (H) Bar graph depicting the relative fold change in luciferase reporter activity in SGR-Feo cells transfected with either siNT, siNT plus empty vector (EV-GFP), siSEC16A, or siSEC16A plus SEC16A-GFP. The respective overexpression vectors were transfected at 24 h posttransfection with the respective siRNAs. The luciferase reporter activity was determined 72 h after siRNA transfection. Scale bar, 100 μm. The data presented are means ± the SEM of three independent experiments. Statistical analysis was done using a Student t test (ns, nonsignificant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001).

Article Snippet: Lentivirus particles harboring the respective shRNA were produced in 293FT cells by cotransfection of lentiviral shRNA vector (pLKO.1 puro), pCMVR8.74 (Addgene 22036), and pMD2.G (Addgene 12259) with a Cal-Phos mammalian transfection kit according to the manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, Knockdown, Transduction, Western Blot, Expressing, Control, Transfection, Cell Culture, Immunofluorescence, Over Expression, Plasmid Preparation

Journal: Journal of Virology

Article Title: The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle

doi: 10.1128/jvi.00180-23

Figure Lengend Snippet: ERES and ERGIC role in HCV assembly and secretion. (A) Illustration representing the experimental timeline and approach used to determine the role of ERES, ERGIC, and TFG in HCV assembly and secretion. (B) Western blot analysis depicting the knockdown efficiency of SEC16A, ERGIC-53, and TFG in cells transfected with respective siRNA. β-Actin and GAPDH were used as internal loading controls. (C to F) Bar graphs representing the relative fold change in the intracellular (C) and extracellular (E) HCV genome copies and the relative change in intracellular (D) and extracellular (F) infectious viral titers in HCV-infected cells at 96 hpi and 48 h posttransfection with siRNAs against SEC16A, ERGIC-53, and TFG. (G and H) Bar graphs representing the relative fold change in the level of intra- and extracellular viral RNA levels (G) and infectious virus titers (H) in HCV-infected cells 48 hpi subjected to 24-h treatment with the indicated concentrations of FLI-06. Dimethyl sulfoxide was used as vehicle control (VC). (I) Confocal images representing the ERGIC (green) morphology and HCV envelope (E2, red) distribution in cells treated with DMSO (VC) or 10 μM FLI-06 for 24 h. Scale bar, 10 μm. The data presented are means ± the SEM of three independent experiments. Statistical analysis was done by using a Student t test (ns, nonsignificant; ****, P < 0.0001).

Article Snippet: Lentivirus particles harboring the respective shRNA were produced in 293FT cells by cotransfection of lentiviral shRNA vector (pLKO.1 puro), pCMVR8.74 (Addgene 22036), and pMD2.G (Addgene 12259) with a Cal-Phos mammalian transfection kit according to the manufacturer’s instructions.

Techniques: Western Blot, Knockdown, Transfection, Infection, Virus, Control

Journal: Journal of Virology

Article Title: The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle

doi: 10.1128/jvi.00180-23

Figure Lengend Snippet: SEC16A and TFG overexpression rescue the HCV assembly and release in SEC16A and TFG knockdown cells. (A to D) HCV-infected Huh7 cells at 48 hpi were transfected with siNT, siSEC16A, siTFG siRNAs and 24 h later transfected with the SEC16A-GFP (A and B) or TFG-FLAG (C and D), respectively. siNT-transfected cells were transfected with corresponding empty vectors (EV-GFP or EV) at 24 h posttransfection with the non-targeting siRNA. HCV infectious titers were determined in the culture supernatants, and cells by using an FFU assay. The bar graphs depict the intra- and extracellular viral titers from the HCV-infected Huh7 cells subjected to respective experimental conditions. (E) Western blot analysis and immunofluorescence images depicting the SEC16A knockdown efficiency and overexpression of SEC16A-GFP or EV-GFP. NS5A and GAPDH were used as an HCV infection marker and an internal loading control, respectively. Scale bar, 100 μm. (F) Western blot analysis depicting the TFG knockdown efficiency and overexpression of TFG-FLAG in HCV-infected Huh7 cells. NS3 and GAPDH were used as an HCV infection marker and an internal loading control, respectively.

Article Snippet: Lentivirus particles harboring the respective shRNA were produced in 293FT cells by cotransfection of lentiviral shRNA vector (pLKO.1 puro), pCMVR8.74 (Addgene 22036), and pMD2.G (Addgene 12259) with a Cal-Phos mammalian transfection kit according to the manufacturer’s instructions.

Techniques: Over Expression, Knockdown, Infection, Transfection, Western Blot, Immunofluorescence, Marker, Control