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1.
FIG. 4.

FIG. 4. From: Superinfection Exclusion in Cells Infected with Hepatitis C Virus .

JFH-SGneo cells exhibit a postentry superinfection block. (A) Huh-7.5 and JFH-SGneo cells were infected with HCVcc-Rluc. At each time point, luciferase activity was determined from triplicate wells and normalized to the average cellular ATP content for each cell type at that time point. Each bar is the average value of triplicate wells; error bars show standard deviations. (B and C) Huh-7.5 cells (B) or BILN 2061-cured JFH-SGneo (C) and JFH-SGneo cells were infected with HCVcc-Rluc in the presence of 2′-C-methyladenosine. At each time point, luciferase activity was determined from triplicate wells and normalized to the average cellular ATP content for each cell type at that time point. Each bar is the average value of triplicate wells; error bars show standard deviations.

Donna M. Tscherne, et al. J Virol. 2007 April;81(8):3693-3703.
2.
FIG. 6.

FIG. 6. From: Superinfection Exclusion in Cells Infected with Hepatitis C Virus .

Ectopic expression of CD81 renders stable J6/JFH-FLneo replicon cells permissive for HCVpp infection. (A) Transduction of J6/JFH-FLneo cells with TRIP-CD81 restores CD81 expression. J6/JFH-FLneo (black lines), J6/JFH-FLneo-CD81 (shaded area), and Huh-7.5 (gray lines) cells were surface stained for CD81 and analyzed by flow cytometry. Dashed lines indicate the IgG isotype controls. Max, maximum. (B) Huh-7.5, J6/JFH-FLneo, J6/JFH-FLneo-CD81, and J6/JFH-FLneo-CD9 cells transduced with TRIP-CD9 were infected with HCVpp-Luc (genotype 1a strain H77) or VSVGpp-Luc, and luciferase activity was measured at 72 hpi. HCVpp-Luc RLU were normalized to VSVGpp-Luc RLU. Each bar is the average value of triplicate wells; error bars show the standard deviations.

Donna M. Tscherne, et al. J Virol. 2007 April;81(8):3693-3703.
3.
FIG. 7.

FIG. 7. From: Superinfection Exclusion in Cells Infected with Hepatitis C Virus .

BILN 2061-treated, J6/JFH-FLneo replicon-containing cells have increased CD81 surface expression and are permissive for HCVpp infection. (A) J6/JFH-FLneo and Huh-7.5 parental cells were treated with BILN 2061 or DMSO for a period of 23 days. At 4 days (4d), 9 days (9d), and 23 days (23d) posttreatment, DMSO- and BILN 2061-treated cells were challenged with HCVpp-Luc or VSVGpp-Luc. Luciferase activity was measured at 72 hpi, and HCVpp-Luc RLU were normalized to VSVGpp-Luc RLU. Each bar, expressed as the percentage of DMSO-treated Huh-7.5 RLU, is the average value of triplicate wells; error bars show the standard deviations. (B) At 23 days posttreatment, BILN 2061 (dashed, black line)- and DMSO (black line)-treated J6/JFH-FLneo and Huh-7.5 (gray line) cells were immunostained for CD81 surface expression and analyzed by flow cytometry. Max, maximum.

Donna M. Tscherne, et al. J Virol. 2007 April;81(8):3693-3703.
4.
FIG. 1.

FIG. 1. From: Superinfection Exclusion in Cells Infected with Hepatitis C Virus .

Cells harboring HCV do not support HCVcc superinfection. (A) Huh-7.5 cells were infected with J6/JFH HCVcc and maintained in parallel with naïve cells. At 4 dpi, when approximately 77% of cells were NS5A positive, cells acutely infected with J6/JFH and naïve cells were superinfected with HCVcc-Rluc. Each bar, expressed as the percentage of the Huh-7.5 cell RLU, is the average value of triplicate wells; error bars show the standard deviations. RLU, relative light units. (B) Huh-7.5 cells and HCV full-length or subgenomic replicon-containing cells were infected with HCVcc-Rluc. Luciferase activity was measured at 24 hpi. Values, expressed as the percentage of Huh-7.5 RLU, are the combined data from two independent experiments done in triplicate; error bars represent the standard errors of the means. (C) Huh-7.5 cells and HCV replicon-containing cells were infected with VSV and overlaid with agarose. After 16 h of incubation, cells were fixed with formaldehyde and stained with crystal violet to visualize plaques. Each bar is the mean titer from duplicate wells; error bars represent the standard errors of the means.

Donna M. Tscherne, et al. J Virol. 2007 April;81(8):3693-3703.
5.
FIG. 5.

FIG. 5. From: Superinfection Exclusion in Cells Infected with Hepatitis C Virus .

Stable J6/JFH-FLneo replicon-containing cells have decreased CD81 protein and RNA levels. (A and B) Huh-7.5 (gray line), JFH-SGneo (black line in panel A), and J6/JFH-FLneo (black line in panel B) cells were surface stained for CD81 and analyzed by flow cytometry. Dashed gray and black lines indicate the IgG isotype controls for Huh-7.5 cells and the JFH-SGneo (A) or J6/JFH-FLneo cells (B), respectively. Max, maximum. (C) Permeabilized Huh-7.5 (gray line) and J6/JFH-FLneo (black line) cells were stained for total cell CD81 protein expression and analyzed by flow cytometry. Dashed gray and black lines indicate the IgG isotype controls for Huh-7.5 and J6/JFH-FLneo cells, respectively. (D) Western blot for CD81 (α-CD81) or actin (α-actin) protein expression in lysates from J6/JFH-FLneo, Huh-7.5, and JFH-SGneo cells. (E) CD81 RNA was amplified using real-time quantitative RT-PCR from total RNA derived from Huh-7.5, J6/JFH-FLneo, and JFH-SGneo cells. Values were normalized to GAPDH RNA levels for each sample and are expressed as relative levels compared to those in Huh-7.5 cells. Values are the combined data from three independent experiments done in triplicate; error bars show standard errors of the means.

Donna M. Tscherne, et al. J Virol. 2007 April;81(8):3693-3703.
6.
FIG. 3.

FIG. 3. From: Superinfection Exclusion in Cells Infected with Hepatitis C Virus .

Cells acutely infected with J6/JFH, but not cells harboring a stable J6/JFH-FLneo replicon, are permissive for HCVpp infection. (A) Huh-7.5 cells were infected with J6/JFH HCVcc and maintained in parallel with naïve cells. At 4 dpi, when approximately 77% of cells were NS5A positive, cells acutely infected with J6/JFH and naïve cells were superinfected with HCVpp-Luc-bearing strain J6 glycoproteins. Each bar, expressed as the percentage of the Huh-7.5 RLU, is the average value of triplicate wells; error bars show the standard deviations. (B) Huh-7.5 cells and HCV full-length or subgenomic replicon-containing cells were infected with HCVpp-Luc bearing HCV genotype 1a strain H77 (black bars) or genotype 2a strain J6 (white bars) glycoproteins or with VSVGpp-Luc. Luciferase activity was measured at 72 hpi. HCVpp-Luc RLU were normalized to VSVGpp-Luc RLU and expressed as a percentage of the Huh-7.5 cell RLU. Each bar shows the combined data from at least two independent experiments done in triplicate; error bars represent the standard errors of the means.

Donna M. Tscherne, et al. J Virol. 2007 April;81(8):3693-3703.
7.
FIG. 2.

FIG. 2. From: Superinfection Exclusion in Cells Infected with Hepatitis C Virus .

HCV subgenomic replicon-containing cells treated with an HCV-specific protease inhibitor become permissive for HCVcc infection. (A) Huh-7.5, JFH-SGneo, Con1-SGneo, and H-SGneo cells were treated with BILN 2061 or DMSO in the absence of G418 for 4 days (4d) or 9 days (9d). BILN 2061- or DMSO-treated cells were infected with HCVcc-Rluc in the absence of inhibitor, and samples were harvested for luciferase assays at 24 hpi. Black bars (DMSO-treated cells) represent the average RLU value over days 4 and 9 for each DMSO-treated cell population compared to that in Huh-7.5 cells. RLU values from BILN 2061-treated cells (gray and white bars) on day 4 or 9 are expressed as the percentage of the DMSO-treated Huh-7.5 RLU on day 4 or 9, respectively. Each bar is the average value of triplicate wells; error bars show the standard deviations. The data shown are representative of at least two independent experiments. (B) HCV RNA levels in DMSO- or BILN 2061-treated JFH-SGneo, Con1-SGneo, and H-SGneo replicon-containing cells at 4 (gray bars) or 9 days (white bars) posttreatment were determined by real-time quantitative RT-PCR. Black bars (DMSO-treated cells) represent the average RNA level over days 4 and 9 for each DMSO-treated cell population. Error bars show the standard deviations.

Donna M. Tscherne, et al. J Virol. 2007 April;81(8):3693-3703.
8.
FIG. 8.

FIG. 8. From: Superinfection Exclusion in Cells Infected with Hepatitis C Virus .

J6/JFH infection of Huh-7.5 cells promotes the emergence of a CD81low cell population. Huh-7.5 cells were infected with HCVcc (J6/JFH) at an MOI of 0.3 and maintained in parallel with naïve cells. (A) At 1, 2, 3, 4, 6, and 8 dpi with J6/JFH, the infected and naïve cell populations were infected with HCVpp-GFP or VSVGpp-GFP. Cells were harvested at 48 hpi. Cells were stained for NS5A and analyzed by flow cytometry for GFP and NS5A expression. The total number of GFP-positive cells resulting from HCVpp-GFP infection was normalized to the total number of GFP-positive cells in the VSVGpp-GFP-infected samples. Values are expressed as a percentage of GFP-positive cells in the naïve population. (B) The J6/JFH-infected cell population, which was infected with HCVpp-GFP and stained for NS5A in panel A, was grouped into NS5A-positive and NS5A-negative cells. GFP expression in NS5A-positive and NS5A-negative cells was determined and plotted as a percentage of the total GFP-positive cells. (C) The J6/JFH-infected and naïve cell populations were analyzed for CD81 surface expression and NS5A expression using immunostaining and flow cytometry at 3, 4, 6, 8, and 17 dpi with J6/JFH. Cells were gated on NS5A-positive (black line) or NS5A-negative (dashed, black line) cells, and CD81 expression was compared to the naïve population (shaded area). The day postinfection (dpi) and percentage of NS5A-positive cells at each time point are indicated above the plots. 3d, 3 days; Max, maximum.

Donna M. Tscherne, et al. J Virol. 2007 April;81(8):3693-3703.

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