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NK cells release soluble factor(s) to suppress expression of <t>HCV</t> <t>RNA</t> and NS5A protein in human hepatocytes. (a) Co-culture of NK cells with HCV JFH1-infected Huh7.5.1 cells. At day 4 postinfection, Huh7.5.1 cells in the low compartment of a 24-well transwell co-culture system were co-cultured with primary NK (PNK) or NK92 cells that were added to the top compartment of the co-culture system. (b) Effect of NK SN on HCV JFH1 RNA expression. JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in media conditioned with or without NK SN (PNK or NK92) for 48 h. Total cellular RNA extracted from the hepatic cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative to the control (% of control, no NK cells on the top compartment or without treatment with NK SN, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments (*, P
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1) Product Images from "Natural killer cells suppress full cycle HCV infection of human hepatocytes"

Article Title: Natural killer cells suppress full cycle HCV infection of human hepatocytes

Journal: Journal of viral hepatitis

doi: 10.1111/j.1365-2893.2008.01014.x

NK cells release soluble factor(s) to suppress expression of HCV RNA and NS5A protein in human hepatocytes. (a) Co-culture of NK cells with HCV JFH1-infected Huh7.5.1 cells. At day 4 postinfection, Huh7.5.1 cells in the low compartment of a 24-well transwell co-culture system were co-cultured with primary NK (PNK) or NK92 cells that were added to the top compartment of the co-culture system. (b) Effect of NK SN on HCV JFH1 RNA expression. JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in media conditioned with or without NK SN (PNK or NK92) for 48 h. Total cellular RNA extracted from the hepatic cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative to the control (% of control, no NK cells on the top compartment or without treatment with NK SN, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments (*, P
Figure Legend Snippet: NK cells release soluble factor(s) to suppress expression of HCV RNA and NS5A protein in human hepatocytes. (a) Co-culture of NK cells with HCV JFH1-infected Huh7.5.1 cells. At day 4 postinfection, Huh7.5.1 cells in the low compartment of a 24-well transwell co-culture system were co-cultured with primary NK (PNK) or NK92 cells that were added to the top compartment of the co-culture system. (b) Effect of NK SN on HCV JFH1 RNA expression. JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in media conditioned with or without NK SN (PNK or NK92) for 48 h. Total cellular RNA extracted from the hepatic cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative to the control (% of control, no NK cells on the top compartment or without treatment with NK SN, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments (*, P

Techniques Used: Expressing, Co-Culture Assay, Infection, Cell Culture, RNA Expression, Quantitative RT-PCR

Time course of inhibition of HCV RNA expression by NK SN. Primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected hepatocyte cultures at day 4 postinfection. Intracellular (a) or extracellular (b) RNA was extracted from hepatocytes or culture supernatants (SN) at indicated time points and subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as either HCV RNA 10 7 copies/ μ g cellular RNA or 10 7 copies/mL SN, respectively. The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P
Figure Legend Snippet: Time course of inhibition of HCV RNA expression by NK SN. Primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected hepatocyte cultures at day 4 postinfection. Intracellular (a) or extracellular (b) RNA was extracted from hepatocytes or culture supernatants (SN) at indicated time points and subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as either HCV RNA 10 7 copies/ μ g cellular RNA or 10 7 copies/mL SN, respectively. The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

Techniques Used: Inhibition, RNA Expression, Infection, Quantitative RT-PCR

Suppression of HCV RNA expression by NK SN under different conditions. Huh7.5.1 cells were cultured in media conditioned with or without primary NK (PNK) SN or NK92 SN for either 24 h prior to HCV infection, or simultaneously or 8 h postinfection. The cells were then washed five times to remove input HCV after 6 h incubation with HCV JFH1 and then cultured in the presence or absence of NK SN for 8 days. Intracellular (a) and extracellular (b) RNA extracted from hepatocytes or culture supernatants (SN) at day 8 postinfection was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as HCV RNA levels relative (%) to the control (untreated cell cultures, which are defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P
Figure Legend Snippet: Suppression of HCV RNA expression by NK SN under different conditions. Huh7.5.1 cells were cultured in media conditioned with or without primary NK (PNK) SN or NK92 SN for either 24 h prior to HCV infection, or simultaneously or 8 h postinfection. The cells were then washed five times to remove input HCV after 6 h incubation with HCV JFH1 and then cultured in the presence or absence of NK SN for 8 days. Intracellular (a) and extracellular (b) RNA extracted from hepatocytes or culture supernatants (SN) at day 8 postinfection was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as HCV RNA levels relative (%) to the control (untreated cell cultures, which are defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

Techniques Used: RNA Expression, Cell Culture, Infection, Incubation, Quantitative RT-PCR

IFN- γ is involved in NK SN-mediated anti-HCV activity. (a) IFN- γ production by primary NK (PNK) cells and NK92 cells. IFN- γ levels in media from PNK cells (five donors) and NK-92 cells were determined by ELISA. (b) HCV JFH1-infected Huh7.5.1 cells were cultured in the presence or absence of NK SN and/or antibody (Ab) against IFN- γ . For the cultures using both NK SN and Ab to IFN- γ , the NK SN was preincubated with the Ab to IFN- γ for 30 min before being added to JFH1-infected hepatocyte cultures at day 4 postinfection. IFN- γ (1000 U/mL) alone was added to the cell cultures as a positive control to determine the neutralization ability of the Ab to IFN- γ . Mouse IgG 2A was used to determine the specificity of the Ab to IFN- γ . Total cellular RNA extracted from hepatocytes was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification at 48 h postexposure to NK SN. The data are expressed as HCV RNA levels relative (% of control, no Abs treatment and no NK SN added, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P
Figure Legend Snippet: IFN- γ is involved in NK SN-mediated anti-HCV activity. (a) IFN- γ production by primary NK (PNK) cells and NK92 cells. IFN- γ levels in media from PNK cells (five donors) and NK-92 cells were determined by ELISA. (b) HCV JFH1-infected Huh7.5.1 cells were cultured in the presence or absence of NK SN and/or antibody (Ab) against IFN- γ . For the cultures using both NK SN and Ab to IFN- γ , the NK SN was preincubated with the Ab to IFN- γ for 30 min before being added to JFH1-infected hepatocyte cultures at day 4 postinfection. IFN- γ (1000 U/mL) alone was added to the cell cultures as a positive control to determine the neutralization ability of the Ab to IFN- γ . Mouse IgG 2A was used to determine the specificity of the Ab to IFN- γ . Total cellular RNA extracted from hepatocytes was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification at 48 h postexposure to NK SN. The data are expressed as HCV RNA levels relative (% of control, no Abs treatment and no NK SN added, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Cell Culture, Positive Control, Neutralization, Quantitative RT-PCR

Effect of NK SN on the expression of IFN- α/β in human hepatocytes. (a and c) IFN- α/β RNA expression. At day 4 postinfection, primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected Huh7.5.1 cells (25%, v/v). Total cellular RNA was extracted from the cell cultures at 12 h posttreatment with NK SN and then subjected to real-time RT-PCR for IFN- α/β and GAPDH RNA quantification. The data were expressed as IFN- α/β RNA levels relative (fold) to the control (without NK SN, which is defined as 1). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments. (b) IFN- α protein expression. HCV JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in the presence or absence NK (PNK and NK-92) SN (25%, v/v) for 48 h. SNs from the cell cultures were then harvested for IFN- α protein ELISA assay. The results shown are mean ± SD of triplicate cultures (*, P
Figure Legend Snippet: Effect of NK SN on the expression of IFN- α/β in human hepatocytes. (a and c) IFN- α/β RNA expression. At day 4 postinfection, primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected Huh7.5.1 cells (25%, v/v). Total cellular RNA was extracted from the cell cultures at 12 h posttreatment with NK SN and then subjected to real-time RT-PCR for IFN- α/β and GAPDH RNA quantification. The data were expressed as IFN- α/β RNA levels relative (fold) to the control (without NK SN, which is defined as 1). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments. (b) IFN- α protein expression. HCV JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in the presence or absence NK (PNK and NK-92) SN (25%, v/v) for 48 h. SNs from the cell cultures were then harvested for IFN- α protein ELISA assay. The results shown are mean ± SD of triplicate cultures (*, P

Techniques Used: Expressing, RNA Expression, Infection, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay

2) Product Images from "Natural killer cells suppress full cycle HCV infection of human hepatocytes"

Article Title: Natural killer cells suppress full cycle HCV infection of human hepatocytes

Journal: Journal of viral hepatitis

doi: 10.1111/j.1365-2893.2008.01014.x

NK cells release soluble factor(s) to suppress expression of HCV RNA and NS5A protein in human hepatocytes. (a) Co-culture of NK cells with HCV JFH1-infected Huh7.5.1 cells. At day 4 postinfection, Huh7.5.1 cells in the low compartment of a 24-well transwell co-culture system were co-cultured with primary NK (PNK) or NK92 cells that were added to the top compartment of the co-culture system. (b) Effect of NK SN on HCV JFH1 RNA expression. JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in media conditioned with or without NK SN (PNK or NK92) for 48 h. Total cellular RNA extracted from the hepatic cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative to the control (% of control, no NK cells on the top compartment or without treatment with NK SN, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments (*, P
Figure Legend Snippet: NK cells release soluble factor(s) to suppress expression of HCV RNA and NS5A protein in human hepatocytes. (a) Co-culture of NK cells with HCV JFH1-infected Huh7.5.1 cells. At day 4 postinfection, Huh7.5.1 cells in the low compartment of a 24-well transwell co-culture system were co-cultured with primary NK (PNK) or NK92 cells that were added to the top compartment of the co-culture system. (b) Effect of NK SN on HCV JFH1 RNA expression. JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in media conditioned with or without NK SN (PNK or NK92) for 48 h. Total cellular RNA extracted from the hepatic cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative to the control (% of control, no NK cells on the top compartment or without treatment with NK SN, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments (*, P

Techniques Used: Expressing, Co-Culture Assay, Infection, Cell Culture, RNA Expression, Quantitative RT-PCR

Time course of inhibition of HCV RNA expression by NK SN. Primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected hepatocyte cultures at day 4 postinfection. Intracellular (a) or extracellular (b) RNA was extracted from hepatocytes or culture supernatants (SN) at indicated time points and subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as either HCV RNA 10 7 copies/ μ g cellular RNA or 10 7 copies/mL SN, respectively. The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P
Figure Legend Snippet: Time course of inhibition of HCV RNA expression by NK SN. Primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected hepatocyte cultures at day 4 postinfection. Intracellular (a) or extracellular (b) RNA was extracted from hepatocytes or culture supernatants (SN) at indicated time points and subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as either HCV RNA 10 7 copies/ μ g cellular RNA or 10 7 copies/mL SN, respectively. The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

Techniques Used: Inhibition, RNA Expression, Infection, Quantitative RT-PCR

Suppression of HCV RNA expression by NK SN under different conditions. Huh7.5.1 cells were cultured in media conditioned with or without primary NK (PNK) SN or NK92 SN for either 24 h prior to HCV infection, or simultaneously or 8 h postinfection. The cells were then washed five times to remove input HCV after 6 h incubation with HCV JFH1 and then cultured in the presence or absence of NK SN for 8 days. Intracellular (a) and extracellular (b) RNA extracted from hepatocytes or culture supernatants (SN) at day 8 postinfection was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as HCV RNA levels relative (%) to the control (untreated cell cultures, which are defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P
Figure Legend Snippet: Suppression of HCV RNA expression by NK SN under different conditions. Huh7.5.1 cells were cultured in media conditioned with or without primary NK (PNK) SN or NK92 SN for either 24 h prior to HCV infection, or simultaneously or 8 h postinfection. The cells were then washed five times to remove input HCV after 6 h incubation with HCV JFH1 and then cultured in the presence or absence of NK SN for 8 days. Intracellular (a) and extracellular (b) RNA extracted from hepatocytes or culture supernatants (SN) at day 8 postinfection was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as HCV RNA levels relative (%) to the control (untreated cell cultures, which are defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

Techniques Used: RNA Expression, Cell Culture, Infection, Incubation, Quantitative RT-PCR

IFN- γ is involved in NK SN-mediated anti-HCV activity. (a) IFN- γ production by primary NK (PNK) cells and NK92 cells. IFN- γ levels in media from PNK cells (five donors) and NK-92 cells were determined by ELISA. (b) HCV JFH1-infected Huh7.5.1 cells were cultured in the presence or absence of NK SN and/or antibody (Ab) against IFN- γ . For the cultures using both NK SN and Ab to IFN- γ , the NK SN was preincubated with the Ab to IFN- γ for 30 min before being added to JFH1-infected hepatocyte cultures at day 4 postinfection. IFN- γ (1000 U/mL) alone was added to the cell cultures as a positive control to determine the neutralization ability of the Ab to IFN- γ . Mouse IgG 2A was used to determine the specificity of the Ab to IFN- γ . Total cellular RNA extracted from hepatocytes was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification at 48 h postexposure to NK SN. The data are expressed as HCV RNA levels relative (% of control, no Abs treatment and no NK SN added, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P
Figure Legend Snippet: IFN- γ is involved in NK SN-mediated anti-HCV activity. (a) IFN- γ production by primary NK (PNK) cells and NK92 cells. IFN- γ levels in media from PNK cells (five donors) and NK-92 cells were determined by ELISA. (b) HCV JFH1-infected Huh7.5.1 cells were cultured in the presence or absence of NK SN and/or antibody (Ab) against IFN- γ . For the cultures using both NK SN and Ab to IFN- γ , the NK SN was preincubated with the Ab to IFN- γ for 30 min before being added to JFH1-infected hepatocyte cultures at day 4 postinfection. IFN- γ (1000 U/mL) alone was added to the cell cultures as a positive control to determine the neutralization ability of the Ab to IFN- γ . Mouse IgG 2A was used to determine the specificity of the Ab to IFN- γ . Total cellular RNA extracted from hepatocytes was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification at 48 h postexposure to NK SN. The data are expressed as HCV RNA levels relative (% of control, no Abs treatment and no NK SN added, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Cell Culture, Positive Control, Neutralization, Quantitative RT-PCR

Effect of NK SN on the expression of IFN- α/β in human hepatocytes. (a and c) IFN- α/β RNA expression. At day 4 postinfection, primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected Huh7.5.1 cells (25%, v/v). Total cellular RNA was extracted from the cell cultures at 12 h posttreatment with NK SN and then subjected to real-time RT-PCR for IFN- α/β and GAPDH RNA quantification. The data were expressed as IFN- α/β RNA levels relative (fold) to the control (without NK SN, which is defined as 1). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments. (b) IFN- α protein expression. HCV JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in the presence or absence NK (PNK and NK-92) SN (25%, v/v) for 48 h. SNs from the cell cultures were then harvested for IFN- α protein ELISA assay. The results shown are mean ± SD of triplicate cultures (*, P
Figure Legend Snippet: Effect of NK SN on the expression of IFN- α/β in human hepatocytes. (a and c) IFN- α/β RNA expression. At day 4 postinfection, primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected Huh7.5.1 cells (25%, v/v). Total cellular RNA was extracted from the cell cultures at 12 h posttreatment with NK SN and then subjected to real-time RT-PCR for IFN- α/β and GAPDH RNA quantification. The data were expressed as IFN- α/β RNA levels relative (fold) to the control (without NK SN, which is defined as 1). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments. (b) IFN- α protein expression. HCV JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in the presence or absence NK (PNK and NK-92) SN (25%, v/v) for 48 h. SNs from the cell cultures were then harvested for IFN- α protein ELISA assay. The results shown are mean ± SD of triplicate cultures (*, P

Techniques Used: Expressing, RNA Expression, Infection, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay

3) Product Images from "Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation"

Article Title: Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation

Journal: Nature medicine

doi: 10.1038/nm.3610

Lipid peroxidation reduces HCV-induced membranous web abundance and alters the EC 50 of DAAs. ( a ) Transmission electron microscopic images of the membranous web in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with DMSO, SKI (1 μM), VE (1 μM), LA (50 μM), or CuOH (10 μM). Scale bar = 500 nm. ( b ) Quantitation of area occupied by double-membrane vesicles (DMV) within individual cells infected with H77S.3 virus and treated with SKI, VE, or LA as in a . * P ≤ 0.002 vs. DMSO by two-sided Mann-Whitney test. ( c ) SKI and VE mask the antiviral effect of PSI-6130 against H77S.3/GLuc replication. (left) Huh-7.5 cells electroporated with H77S.3/GLuc or HJ3-5/GLuc RNA were cultured for 7 d, then treated with DMSO, 10 μM PSI-6130, 1 μM VE or both PSI-6130 and VE. Culture supernatant fluids were replaced every 24 h and assayed for GLuc activity. (right) Huh-7.5 cells were electroporated with H77S.3 RNA, cultured for 5 d and then treated with DMSO, PSI-6130, SKI or VE, or PSI-6130 plus SKI or VE. Cell-associated HCV RNA was quantified by qRT-PCR. Results represent mean ± s.e.m from two (left) or three (right) replicate cultures. ( d ) Inhibition of H77S.3 (left) and HJ3-5 (right) replication by the NS5B inhibitor PSI-6130 in the presence of SKI or VE (each 1 μM) or DMSO vehicle. Inhibition was assessed by quantifying GLuc secreted 48–72 h after drug addition. Results represent mean ± s.e.m. of two replicate cultures. ( e,f ) Inhibition of H77S.3 replication by ( e ) MK-7009, an NS3/4A inhibitor, and ( f ) SCY-635, a host-targeting cyclophilin inhibitor. ( g ) EC 50 values of representative direct- versus indirect-acting antivirals against H77S.3 (left) and HJ3-5 (right) viruses in the presence of SKI or VE (each 1 μM). Assays were carried out as in panels d–f. Colored bars represent limits of the 95% c.i. of EC 50 values calculated from Hill plots. ‘NA’ = not measureable due to poor antiviral activity. See Supplementary Fig. 9 for additional details.
Figure Legend Snippet: Lipid peroxidation reduces HCV-induced membranous web abundance and alters the EC 50 of DAAs. ( a ) Transmission electron microscopic images of the membranous web in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with DMSO, SKI (1 μM), VE (1 μM), LA (50 μM), or CuOH (10 μM). Scale bar = 500 nm. ( b ) Quantitation of area occupied by double-membrane vesicles (DMV) within individual cells infected with H77S.3 virus and treated with SKI, VE, or LA as in a . * P ≤ 0.002 vs. DMSO by two-sided Mann-Whitney test. ( c ) SKI and VE mask the antiviral effect of PSI-6130 against H77S.3/GLuc replication. (left) Huh-7.5 cells electroporated with H77S.3/GLuc or HJ3-5/GLuc RNA were cultured for 7 d, then treated with DMSO, 10 μM PSI-6130, 1 μM VE or both PSI-6130 and VE. Culture supernatant fluids were replaced every 24 h and assayed for GLuc activity. (right) Huh-7.5 cells were electroporated with H77S.3 RNA, cultured for 5 d and then treated with DMSO, PSI-6130, SKI or VE, or PSI-6130 plus SKI or VE. Cell-associated HCV RNA was quantified by qRT-PCR. Results represent mean ± s.e.m from two (left) or three (right) replicate cultures. ( d ) Inhibition of H77S.3 (left) and HJ3-5 (right) replication by the NS5B inhibitor PSI-6130 in the presence of SKI or VE (each 1 μM) or DMSO vehicle. Inhibition was assessed by quantifying GLuc secreted 48–72 h after drug addition. Results represent mean ± s.e.m. of two replicate cultures. ( e,f ) Inhibition of H77S.3 replication by ( e ) MK-7009, an NS3/4A inhibitor, and ( f ) SCY-635, a host-targeting cyclophilin inhibitor. ( g ) EC 50 values of representative direct- versus indirect-acting antivirals against H77S.3 (left) and HJ3-5 (right) viruses in the presence of SKI or VE (each 1 μM). Assays were carried out as in panels d–f. Colored bars represent limits of the 95% c.i. of EC 50 values calculated from Hill plots. ‘NA’ = not measureable due to poor antiviral activity. See Supplementary Fig. 9 for additional details.

Techniques Used: Transmission Assay, Quantitation Assay, Infection, MANN-WHITNEY, Cell Culture, Activity Assay, Quantitative RT-PCR, Inhibition

Inhibition of lipid peroxidation by SKI or VE promotes production and spread of infectious genotype 1 HCV. ( a ) Infectious virus yields from Huh-7.5 cells transfected with the indicated viral RNAs and grown in the presence of 1 μM SKI, 1 μM VE or DMSO vehicle. Culture supernatant fluids were harvested and replaced with fresh media containing compounds every 24 h. Infectivity titers are expressed as focus forming units (FFU) ml −1 . Data shown are mean ± s.e.m. from three replicate cultures. ( b ) SKI promotes spread of H77S.3 but inhibits HJ3-5 virus. Cells electroporated with H77S.3 or HJ3-5 RNAs were mixed with carboxyfluorescein succinimidyl ester (CFSE)-labeled naïve Huh-7.5 cells; CFSE/NS5A double-positive cells (upper right quadrant) are indicative of virus spread. ( c ) Buoyant density of H77S.3 virus particles released from H77S.3 RNA-transfected Huh-7.5 cells grown in 1 μM SKI (left) or 1 μM VE (right) vs. DMSO control. Fractions from isopycnic iodixanol gradients were assayed for infectious virus (FFU) or HCV RNA (GE = genome equivalents). The data shown are representative of two independent experiments.
Figure Legend Snippet: Inhibition of lipid peroxidation by SKI or VE promotes production and spread of infectious genotype 1 HCV. ( a ) Infectious virus yields from Huh-7.5 cells transfected with the indicated viral RNAs and grown in the presence of 1 μM SKI, 1 μM VE or DMSO vehicle. Culture supernatant fluids were harvested and replaced with fresh media containing compounds every 24 h. Infectivity titers are expressed as focus forming units (FFU) ml −1 . Data shown are mean ± s.e.m. from three replicate cultures. ( b ) SKI promotes spread of H77S.3 but inhibits HJ3-5 virus. Cells electroporated with H77S.3 or HJ3-5 RNAs were mixed with carboxyfluorescein succinimidyl ester (CFSE)-labeled naïve Huh-7.5 cells; CFSE/NS5A double-positive cells (upper right quadrant) are indicative of virus spread. ( c ) Buoyant density of H77S.3 virus particles released from H77S.3 RNA-transfected Huh-7.5 cells grown in 1 μM SKI (left) or 1 μM VE (right) vs. DMSO control. Fractions from isopycnic iodixanol gradients were assayed for infectious virus (FFU) or HCV RNA (GE = genome equivalents). The data shown are representative of two independent experiments.

Techniques Used: Inhibition, Transfection, Infection, Labeling

Lipid peroxidation regulates wild-type HCV replication and represses cell culture-adapted virus in primary human liver cultures. ( a ) Effects of SKI, VE (each 1 μM), LA (20 μM), LA + SKI, LA + VE, or a DAA (MK-0608, 10 μM) on replication of wild-type H77c/GLuc or HCV-N/GLuc RNAs, or a replication-defective control (H77c/GLuc-AAG) in Huh-7.5 cells. ( b ) Wild-type JFH1/GLuc RNA was electroporated and treated with drugs as in ( a ) with PSI-6130 (10 μM) as the DAA control. ( c ) Phase contrast microscopy of fetal hepatoblasts at 3 d. Scale bar, 50 μm. ( d ) Human fetal hepatoblasts (HFH) were infected with H77S.3/GLuc or HJ3-5/GLuc viruses in HFH media containing SKI or VE (each 1 μM), LA (50 μM), LA + VE, or a DAA, MK-0608 or PSI-6130 (each 10 μM) and assayed for GLuc. Results represent mean ± s.e.m. from three replicate cultures with cells from two donors. ( e ) HFH were infected with H77S.3 or HJ3-5 (MOI = 0.01) and treated as in d . Cell-associated viral RNA was quantified by qRT-PCR at 5 d (* P
Figure Legend Snippet: Lipid peroxidation regulates wild-type HCV replication and represses cell culture-adapted virus in primary human liver cultures. ( a ) Effects of SKI, VE (each 1 μM), LA (20 μM), LA + SKI, LA + VE, or a DAA (MK-0608, 10 μM) on replication of wild-type H77c/GLuc or HCV-N/GLuc RNAs, or a replication-defective control (H77c/GLuc-AAG) in Huh-7.5 cells. ( b ) Wild-type JFH1/GLuc RNA was electroporated and treated with drugs as in ( a ) with PSI-6130 (10 μM) as the DAA control. ( c ) Phase contrast microscopy of fetal hepatoblasts at 3 d. Scale bar, 50 μm. ( d ) Human fetal hepatoblasts (HFH) were infected with H77S.3/GLuc or HJ3-5/GLuc viruses in HFH media containing SKI or VE (each 1 μM), LA (50 μM), LA + VE, or a DAA, MK-0608 or PSI-6130 (each 10 μM) and assayed for GLuc. Results represent mean ± s.e.m. from three replicate cultures with cells from two donors. ( e ) HFH were infected with H77S.3 or HJ3-5 (MOI = 0.01) and treated as in d . Cell-associated viral RNA was quantified by qRT-PCR at 5 d (* P

Techniques Used: Cell Culture, Microscopy, Infection, Quantitative RT-PCR

Resistance to lipid peroxidation is tightly linked to robust replication in cell culture. ( a ) (upper panel) Cell culture-adaptive mutations in TNcc 34 (yellow arrowheads) confer resistance to lipid peroxidation when introduced into H77S.3/GLuc IS (H77S.3/GLuc in which the adaptive mutation S2204I has been removed (black arrowhead), see Supplementary Fig. 10b for details). Huh-7.5 cells were treated with DMSO, 1 μM SKI, 1 μM VE, 10 μM CuOH, CuOH plus VE, or 30 μM sofosbuvir (DAA) beginning 6 h following RNA electroporation. GLuc secretion was measured between 48–72 h. (lower panel) TNcc mutations in NS3 (helicase) and NS4B are not required for lipid peroxidation resistance. Combinations of TNcc substitutions were introduced into H77S.3/GLuc IS/GS (NS proteins shown only) that contains the compensatory mutation G1909S (GS) in NS4B (red arrowhead, see Supplementary Fig. 11 ). Data shown represent mean GLuc activity ± s.e.m. from two independent experiments. L.O.D. = limit of detection. ( b ) (top) H77D genome containing the I2204S substitution (black), 8 TNcc-derived mutations (yellow) and 3 novel compensatory mutations (red) in the H77S.3 background. (bottom) Huh-7.5 cells transfected with the indicated RNAs encoding GLuc (left) or lacking GLuc (right) were treated with DMSO or 1 μM VE and secreted GLuc activity (left) or released infectious virus (right) measured between 48–72 h. Data shown represent means ± s.d. from triplicate cultures in a representative experiment. ( c ) Structural models of (left) NS4A and (right) NS5B membrane interactions showing key residues that determine sensitivity to lipid peroxidation. ( d ) EC 50 of direct- vs. indirect-acting antivirals against H77D in the presence of SKI or VE (each 1 μM). Assays were carried out as in Fig. 5g . ( e ) Huh-7.5 cells transfected with H77S.3/GLuc or HJ3-5/GLuc RNA, genome-length JFH-2 RNA, or subgenomic RNAs encoding FLuc (S52 and ED43) were treated with DMSO, 1 μM SKI or VE, 10 uM CuOH or CuOH plus VE. Data shown represent percent GLuc (H77S.3 and HJ3-5), RNA copies (JFH-2) or FLuc activities (S52 and ED43) at 72 h relative to DMSO controls. Data shown represent mean ± s.e.m. from three independent experiments. ( f ) The HCV replicase is uniquely regulated by lipid peroxidation. The impact of SKI or VE (each 1 μM) or LA (50 μM) on the abundance of viral RNA (left) or yields of infectious virus (right) was determined for a panel of RNA viruses following infection of Huh-7.5 cells. In addition to H77S.3, viruses studied included the hepatotropic picornavirus, hepatitis A virus (HAV), representative flaviviruses (dengue virus, DENV; West Nile virus, WNV; yellow fever virus, YFV), alphaviruses (Sindbis virus, SINV; Ross River virus, RRV; Chikungunya virus, CHIKV), and lymphocytic choriomeningitis virus (LCMV). Data shown are mean ± s.e.m. from three replicate cultures. See Supplementary Fig. 12 for additional details.
Figure Legend Snippet: Resistance to lipid peroxidation is tightly linked to robust replication in cell culture. ( a ) (upper panel) Cell culture-adaptive mutations in TNcc 34 (yellow arrowheads) confer resistance to lipid peroxidation when introduced into H77S.3/GLuc IS (H77S.3/GLuc in which the adaptive mutation S2204I has been removed (black arrowhead), see Supplementary Fig. 10b for details). Huh-7.5 cells were treated with DMSO, 1 μM SKI, 1 μM VE, 10 μM CuOH, CuOH plus VE, or 30 μM sofosbuvir (DAA) beginning 6 h following RNA electroporation. GLuc secretion was measured between 48–72 h. (lower panel) TNcc mutations in NS3 (helicase) and NS4B are not required for lipid peroxidation resistance. Combinations of TNcc substitutions were introduced into H77S.3/GLuc IS/GS (NS proteins shown only) that contains the compensatory mutation G1909S (GS) in NS4B (red arrowhead, see Supplementary Fig. 11 ). Data shown represent mean GLuc activity ± s.e.m. from two independent experiments. L.O.D. = limit of detection. ( b ) (top) H77D genome containing the I2204S substitution (black), 8 TNcc-derived mutations (yellow) and 3 novel compensatory mutations (red) in the H77S.3 background. (bottom) Huh-7.5 cells transfected with the indicated RNAs encoding GLuc (left) or lacking GLuc (right) were treated with DMSO or 1 μM VE and secreted GLuc activity (left) or released infectious virus (right) measured between 48–72 h. Data shown represent means ± s.d. from triplicate cultures in a representative experiment. ( c ) Structural models of (left) NS4A and (right) NS5B membrane interactions showing key residues that determine sensitivity to lipid peroxidation. ( d ) EC 50 of direct- vs. indirect-acting antivirals against H77D in the presence of SKI or VE (each 1 μM). Assays were carried out as in Fig. 5g . ( e ) Huh-7.5 cells transfected with H77S.3/GLuc or HJ3-5/GLuc RNA, genome-length JFH-2 RNA, or subgenomic RNAs encoding FLuc (S52 and ED43) were treated with DMSO, 1 μM SKI or VE, 10 uM CuOH or CuOH plus VE. Data shown represent percent GLuc (H77S.3 and HJ3-5), RNA copies (JFH-2) or FLuc activities (S52 and ED43) at 72 h relative to DMSO controls. Data shown represent mean ± s.e.m. from three independent experiments. ( f ) The HCV replicase is uniquely regulated by lipid peroxidation. The impact of SKI or VE (each 1 μM) or LA (50 μM) on the abundance of viral RNA (left) or yields of infectious virus (right) was determined for a panel of RNA viruses following infection of Huh-7.5 cells. In addition to H77S.3, viruses studied included the hepatotropic picornavirus, hepatitis A virus (HAV), representative flaviviruses (dengue virus, DENV; West Nile virus, WNV; yellow fever virus, YFV), alphaviruses (Sindbis virus, SINV; Ross River virus, RRV; Chikungunya virus, CHIKV), and lymphocytic choriomeningitis virus (LCMV). Data shown are mean ± s.e.m. from three replicate cultures. See Supplementary Fig. 12 for additional details.

Techniques Used: Cell Culture, Mutagenesis, Electroporation, Activity Assay, Derivative Assay, Transfection, Infection

SKI enhances genotype 1 HCV replication while suppressing JFH1-based viruses by inhibiting type 2 sphingosine kinase (SPHK2). ( a ) HCV RNA genomes that express Gaussia Luciferase (GLuc) fused to foot-and-mouth disease virus 2A autoprotease as part of the HCV polyprotein. Arrowheads indicate cell culture-adaptive mutations. ( b ) (left) Dose-response effects of SKI on replication of H77S.3/GLuc (red) or HJ3-5/GLuc (blue) RNAs in Huh-7.5 cells. (right) Effect of 1 μM SKI on replication of H77S.3 (red) or HJ3-5 (blue) RNAs. Data shown represent relative amounts of GLuc secreted between 48–72 h (left) or intracellular RNA levels at 72 h (right). ( c ) Effect of 1 μM SKI on GLuc activities of the indicated viruses are presented as fold-change from baseline (6 h). ( d ) Flow cytometric analysis of NS5A expression in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with 1 μM SKI or DMSO. ( e–f ) Effect of siRNAs targeting SPHK isoforms or non-targeting control siRNA on replication of different HCV RNAs ( e ) and abundance of each SPHK isoform ( f ). Results represent the mean ± s.e.m. from two independent ( b,c,d ) or triplicate ( e ) experiments. * P
Figure Legend Snippet: SKI enhances genotype 1 HCV replication while suppressing JFH1-based viruses by inhibiting type 2 sphingosine kinase (SPHK2). ( a ) HCV RNA genomes that express Gaussia Luciferase (GLuc) fused to foot-and-mouth disease virus 2A autoprotease as part of the HCV polyprotein. Arrowheads indicate cell culture-adaptive mutations. ( b ) (left) Dose-response effects of SKI on replication of H77S.3/GLuc (red) or HJ3-5/GLuc (blue) RNAs in Huh-7.5 cells. (right) Effect of 1 μM SKI on replication of H77S.3 (red) or HJ3-5 (blue) RNAs. Data shown represent relative amounts of GLuc secreted between 48–72 h (left) or intracellular RNA levels at 72 h (right). ( c ) Effect of 1 μM SKI on GLuc activities of the indicated viruses are presented as fold-change from baseline (6 h). ( d ) Flow cytometric analysis of NS5A expression in Huh-7.5 cells electroporated with H77S.3 or HJ3-5 RNA and treated with 1 μM SKI or DMSO. ( e–f ) Effect of siRNAs targeting SPHK isoforms or non-targeting control siRNA on replication of different HCV RNAs ( e ) and abundance of each SPHK isoform ( f ). Results represent the mean ± s.e.m. from two independent ( b,c,d ) or triplicate ( e ) experiments. * P

Techniques Used: Luciferase, Cell Culture, Flow Cytometry, Expressing

4) Product Images from "Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture"

Article Title: Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

Journal: Journal of Virology

doi: 10.1128/JVI.77.5.3181-3190.2003

Detection of HCV proteins and RNA in Huh-7.5 cells transiently transfected with subgenomic and full-length HCV RNAs. (Top) Ninety-six hours after RNA transfection of Huh-7.5 cells, the monolayers were labeled with 35 S protein-labeling mixture and lysed, and NS3, NS4B, and NS5A were analyzed by immunoprecipitation, SDS- 10% PAGE, and autoradiography. The positions of the molecular-mass standards are given on the left, and HCV-specific proteins are indicated on the right. (Middle) Total cellular RNA was extracted 96 h posttransfection, and HCV RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA to the pol − negative control is shown (HCV RNA/pol − ). (Bottom) Ninety-six hours after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for HCV NS3, and analyzed by FACS. The percentages of cells expressing NS3 relative to an isotype-matched irrelevant IgG are displayed. Values of
Figure Legend Snippet: Detection of HCV proteins and RNA in Huh-7.5 cells transiently transfected with subgenomic and full-length HCV RNAs. (Top) Ninety-six hours after RNA transfection of Huh-7.5 cells, the monolayers were labeled with 35 S protein-labeling mixture and lysed, and NS3, NS4B, and NS5A were analyzed by immunoprecipitation, SDS- 10% PAGE, and autoradiography. The positions of the molecular-mass standards are given on the left, and HCV-specific proteins are indicated on the right. (Middle) Total cellular RNA was extracted 96 h posttransfection, and HCV RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA to the pol − negative control is shown (HCV RNA/pol − ). (Bottom) Ninety-six hours after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for HCV NS3, and analyzed by FACS. The percentages of cells expressing NS3 relative to an isotype-matched irrelevant IgG are displayed. Values of

Techniques Used: Transfection, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Negative Control, Staining, FACS, Expressing

5) Product Images from "Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication"

Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

Journal: Journal of Virology

doi: 10.1128/JVI.76.24.13001-13014.2002

Effect(s) of alternative substitutions at position 2204 in NS5A on HCV RNA replication. Huh-7.5 cells were transfected with 1 μg of the SG-5′HE replicons carrying the indicated amino acid substitutions and 2 × 10 5 cells plated in 35-mm-diameter wells. After 24, 48, and 96 h in culture, total cellular RNA was extracted and HCV RNA levels were measured as described in Materials and Methods. The ratio of HCV RNA relative to the pol − defective subgenomic RNA (HCV RNA/pol − ) was plotted against the time posttransfection. The increase in HCV RNA above pol − is indicated above each bar. In this figure the levels of HCV RNA relative to the pol − are the highest we have achieved so far. When these RNAs were transfected into Huh-7.5 cells a second time, a similar trend in HCV RNA accumulation was observed.
Figure Legend Snippet: Effect(s) of alternative substitutions at position 2204 in NS5A on HCV RNA replication. Huh-7.5 cells were transfected with 1 μg of the SG-5′HE replicons carrying the indicated amino acid substitutions and 2 × 10 5 cells plated in 35-mm-diameter wells. After 24, 48, and 96 h in culture, total cellular RNA was extracted and HCV RNA levels were measured as described in Materials and Methods. The ratio of HCV RNA relative to the pol − defective subgenomic RNA (HCV RNA/pol − ) was plotted against the time posttransfection. The increase in HCV RNA above pol − is indicated above each bar. In this figure the levels of HCV RNA relative to the pol − are the highest we have achieved so far. When these RNAs were transfected into Huh-7.5 cells a second time, a similar trend in HCV RNA accumulation was observed.

Techniques Used: Transfection

Effect(s) of S2194A and S2194D mutations on HCV RNA replication. S2194 was replaced with Ala or Asp in the selectable bicistronic replicon SG-Neo (S2204I), and RNA was transcribed in vitro. (A) RNA transcripts were transfected into Huh-7 cells, and G418-selected colonies were fixed and stained with crystal violet. The relative G418 transduction efficiencies are indicated below each dish. (B) Ninety-six hours posttransfection Huh-7.5 cells were labeled with [ 35 S]methionine and [ 35 S]cysteine for 10 h. Cells were lysed, and HCV proteins were isolated by immunoprecipitation using a patient serum specific for NS3, NS4B, and NS5A. HCV proteins and the positions of protein molecular weight standards (in thousands) are shown. The ratio of HCV RNA relative to the pol − negative control at 96 h posttransfection is shown below each track (HCV RNA/pol − ). The results illustrated are representative of two independent transfections.
Figure Legend Snippet: Effect(s) of S2194A and S2194D mutations on HCV RNA replication. S2194 was replaced with Ala or Asp in the selectable bicistronic replicon SG-Neo (S2204I), and RNA was transcribed in vitro. (A) RNA transcripts were transfected into Huh-7 cells, and G418-selected colonies were fixed and stained with crystal violet. The relative G418 transduction efficiencies are indicated below each dish. (B) Ninety-six hours posttransfection Huh-7.5 cells were labeled with [ 35 S]methionine and [ 35 S]cysteine for 10 h. Cells were lysed, and HCV proteins were isolated by immunoprecipitation using a patient serum specific for NS3, NS4B, and NS5A. HCV proteins and the positions of protein molecular weight standards (in thousands) are shown. The ratio of HCV RNA relative to the pol − negative control at 96 h posttransfection is shown below each track (HCV RNA/pol − ). The results illustrated are representative of two independent transfections.

Techniques Used: In Vitro, Transfection, Staining, Transduction, Labeling, Isolation, Immunoprecipitation, Molecular Weight, Negative Control

Detection of HCV proteins and RNA in Huh-7.5 and Huh-7 cells transiently transfected with HCV RNA. Top panel, Huh-7.5 and Huh-7 cells were transfected with the subgenomic replicons pol − (lanes 1 and 7), SG-5′HE (S2204I) (lanes 2 and 8), SG-5′HE (5AΔ47) (lanes 3 and 9), SG-Neo (S2204I) (lanes 4 and 10), SG-Neo (5AΔ47) (lanes 5 and 11), and FL (S2204I) HCV RNA (lanes 6 and 12). At 96 h posttransfection, monolayers were incubated for 10 h in the presence of [ 35 S]methionine and [ 35 S]cysteine. Labeled cells were lysed and immunoprecipitated with HCV-positive human serum (anti-NS3, NS4B, and NS5A), and labeled proteins were separated by SDS-10% PAGE. Note that twice the amount of immunoprecipitated sample was loaded in lanes 6 and 12. The mobilities of molecular weight standards (in thousands) are indicated on the left, and the migration of NS3, NS4B, NS5A, and 5AΔ47 is shown on the right. For data in the panel directly below the gel, total cellular RNA was extracted at 96 h posttransfection and quantified for HCV RNA levels as described in the Materials and Methods. The ratio of HCV RNA relative to the pol − defective replicon is shown (HCV RNA/pol − ). HCV RNA levels relative to the pol − control were comparable in three independent experiments. For data in the bottom two panels, 96 h after transfection cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for either HCV core or NS3 antigens, and analyzed by FACS. The percentage of cells expressing core and NS3 relative to an isotype matched irrelevant IgG is displayed. Values
Figure Legend Snippet: Detection of HCV proteins and RNA in Huh-7.5 and Huh-7 cells transiently transfected with HCV RNA. Top panel, Huh-7.5 and Huh-7 cells were transfected with the subgenomic replicons pol − (lanes 1 and 7), SG-5′HE (S2204I) (lanes 2 and 8), SG-5′HE (5AΔ47) (lanes 3 and 9), SG-Neo (S2204I) (lanes 4 and 10), SG-Neo (5AΔ47) (lanes 5 and 11), and FL (S2204I) HCV RNA (lanes 6 and 12). At 96 h posttransfection, monolayers were incubated for 10 h in the presence of [ 35 S]methionine and [ 35 S]cysteine. Labeled cells were lysed and immunoprecipitated with HCV-positive human serum (anti-NS3, NS4B, and NS5A), and labeled proteins were separated by SDS-10% PAGE. Note that twice the amount of immunoprecipitated sample was loaded in lanes 6 and 12. The mobilities of molecular weight standards (in thousands) are indicated on the left, and the migration of NS3, NS4B, NS5A, and 5AΔ47 is shown on the right. For data in the panel directly below the gel, total cellular RNA was extracted at 96 h posttransfection and quantified for HCV RNA levels as described in the Materials and Methods. The ratio of HCV RNA relative to the pol − defective replicon is shown (HCV RNA/pol − ). HCV RNA levels relative to the pol − control were comparable in three independent experiments. For data in the bottom two panels, 96 h after transfection cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for either HCV core or NS3 antigens, and analyzed by FACS. The percentage of cells expressing core and NS3 relative to an isotype matched irrelevant IgG is displayed. Values

Techniques Used: Transfection, Incubation, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Molecular Weight, Migration, Staining, FACS, Expressing

HCV RNA accumulation after transfection of Huh-7.5 cells with full-length HCV RNA. One microgram of in vitro-transcribed RNA was electroporated into Huh-7.5, and 2 × 10 5 cells were plated into 35-mm-diameter wells. Total cellular RNA was isolated at 24, 48, and 96 h posttransfection, and HCV RNA levels were quantified as described in the Materials and Methods. The ratio of HCV RNA relative to the pol − defective subgenomic RNA (HCV RNA/pol − ) was plotted against the time posttransfection, and similar results were obtained when this experiment was repeated
Figure Legend Snippet: HCV RNA accumulation after transfection of Huh-7.5 cells with full-length HCV RNA. One microgram of in vitro-transcribed RNA was electroporated into Huh-7.5, and 2 × 10 5 cells were plated into 35-mm-diameter wells. Total cellular RNA was isolated at 24, 48, and 96 h posttransfection, and HCV RNA levels were quantified as described in the Materials and Methods. The ratio of HCV RNA relative to the pol − defective subgenomic RNA (HCV RNA/pol − ) was plotted against the time posttransfection, and similar results were obtained when this experiment was repeated

Techniques Used: Transfection, In Vitro, Isolation

Effect(s) of combining NS3 and NS5A mutations on HCV RNA replication. Subgenomic replicons lacking neo (SG-5′HE) were generated carrying S2204I with further mutations in NS3. (A) For the gel shown at top, 96 h after RNA transfection of Huh-7.5 cells, monolayers were labeled with 35 S-protein labeling mixture; cells were lysed; and NS3, NS4A, and NS5A were analyzed by immunoprecipitation, SDS-10% PAGE, and autoradiography. Positions of the molecular weight standards (in thousands) are given on the left, and HCV-specific proteins are indicated to the right. For data in the panel directly below the gel, total cellular RNA was extracted at 96 h posttransfection and HCV RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA relative to the pol − negative control is shown (HCV RNA/pol − ). Comparable ratios were obtained in two independent experiments. For data in the bottom two panels, 96 h after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for HCV NS3 and NS5B antigens, and analyzed by FACS. The percentage of cells expressing NS3 and NS5B relative to an isotype-matched irrelevant IgG is displayed. Values
Figure Legend Snippet: Effect(s) of combining NS3 and NS5A mutations on HCV RNA replication. Subgenomic replicons lacking neo (SG-5′HE) were generated carrying S2204I with further mutations in NS3. (A) For the gel shown at top, 96 h after RNA transfection of Huh-7.5 cells, monolayers were labeled with 35 S-protein labeling mixture; cells were lysed; and NS3, NS4A, and NS5A were analyzed by immunoprecipitation, SDS-10% PAGE, and autoradiography. Positions of the molecular weight standards (in thousands) are given on the left, and HCV-specific proteins are indicated to the right. For data in the panel directly below the gel, total cellular RNA was extracted at 96 h posttransfection and HCV RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA relative to the pol − negative control is shown (HCV RNA/pol − ). Comparable ratios were obtained in two independent experiments. For data in the bottom two panels, 96 h after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for HCV NS3 and NS5B antigens, and analyzed by FACS. The percentage of cells expressing NS3 and NS5B relative to an isotype-matched irrelevant IgG is displayed. Values

Techniques Used: Generated, Transfection, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Molecular Weight, Negative Control, Staining, FACS, Expressing

Related Articles

Transfection:

Article Title: Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation
Article Snippet: .. Transfection of wild-type HCV RNA was performed by electroporating 5 μg HCV RNA in 2.5×106 Huh-7.5 cells and seeded into collagen-coated plates (BD Biosciences). ..

Article Title: Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture
Article Snippet: .. The transfected cells were plated in (i) 150-mm-diameter dishes for selection of G418-resistant colonies, (ii) 100-mm-diameter dishes for determining the efficiency of G418-resistant colony formation and fluorescence-activated cell sorting (FACS) analysis, (iii) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, or (iv) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies. .. For dishes requiring G418 selection, 48 h after the cells were plated, the medium was replaced with DMEM- 10% FBS supplemented with 1 mg of G418/ml.

Selection:

Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication
Article Snippet: .. Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection. .. To determine the efficiency of G418-resistant colony formation, transfected cells were plated at multiple densities (between 1 × 103 and 2 × 105 cells), together with cells transfected with pol− RNA transcripts such that the total cell number was maintained at 2 × 105 cells per 100-mm-diameter dish.

Article Title: Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture
Article Snippet: .. The transfected cells were plated in (i) 150-mm-diameter dishes for selection of G418-resistant colonies, (ii) 100-mm-diameter dishes for determining the efficiency of G418-resistant colony formation and fluorescence-activated cell sorting (FACS) analysis, (iii) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, or (iv) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies. .. For dishes requiring G418 selection, 48 h after the cells were plated, the medium was replaced with DMEM- 10% FBS supplemented with 1 mg of G418/ml.

Fluorescence:

Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication
Article Snippet: .. Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection. .. To determine the efficiency of G418-resistant colony formation, transfected cells were plated at multiple densities (between 1 × 103 and 2 × 105 cells), together with cells transfected with pol− RNA transcripts such that the total cell number was maintained at 2 × 105 cells per 100-mm-diameter dish.

Article Title: Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture
Article Snippet: .. The transfected cells were plated in (i) 150-mm-diameter dishes for selection of G418-resistant colonies, (ii) 100-mm-diameter dishes for determining the efficiency of G418-resistant colony formation and fluorescence-activated cell sorting (FACS) analysis, (iii) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, or (iv) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies. .. For dishes requiring G418 selection, 48 h after the cells were plated, the medium was replaced with DMEM- 10% FBS supplemented with 1 mg of G418/ml.

Labeling:

Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication
Article Snippet: .. Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection. .. To determine the efficiency of G418-resistant colony formation, transfected cells were plated at multiple densities (between 1 × 103 and 2 × 105 cells), together with cells transfected with pol− RNA transcripts such that the total cell number was maintained at 2 × 105 cells per 100-mm-diameter dish.

Article Title: Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture
Article Snippet: .. The transfected cells were plated in (i) 150-mm-diameter dishes for selection of G418-resistant colonies, (ii) 100-mm-diameter dishes for determining the efficiency of G418-resistant colony formation and fluorescence-activated cell sorting (FACS) analysis, (iii) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, or (iv) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies. .. For dishes requiring G418 selection, 48 h after the cells were plated, the medium was replaced with DMEM- 10% FBS supplemented with 1 mg of G418/ml.

FACS:

Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication
Article Snippet: .. Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection. .. To determine the efficiency of G418-resistant colony formation, transfected cells were plated at multiple densities (between 1 × 103 and 2 × 105 cells), together with cells transfected with pol− RNA transcripts such that the total cell number was maintained at 2 × 105 cells per 100-mm-diameter dish.

Article Title: Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture
Article Snippet: .. The transfected cells were plated in (i) 150-mm-diameter dishes for selection of G418-resistant colonies, (ii) 100-mm-diameter dishes for determining the efficiency of G418-resistant colony formation and fluorescence-activated cell sorting (FACS) analysis, (iii) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, or (iv) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies. .. For dishes requiring G418 selection, 48 h after the cells were plated, the medium was replaced with DMEM- 10% FBS supplemented with 1 mg of G418/ml.

Immunofluorescence:

Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication
Article Snippet: .. Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection. .. To determine the efficiency of G418-resistant colony formation, transfected cells were plated at multiple densities (between 1 × 103 and 2 × 105 cells), together with cells transfected with pol− RNA transcripts such that the total cell number was maintained at 2 × 105 cells per 100-mm-diameter dish.

Article Title: Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture
Article Snippet: .. The transfected cells were plated in (i) 150-mm-diameter dishes for selection of G418-resistant colonies, (ii) 100-mm-diameter dishes for determining the efficiency of G418-resistant colony formation and fluorescence-activated cell sorting (FACS) analysis, (iii) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, or (iv) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies. .. For dishes requiring G418 selection, 48 h after the cells were plated, the medium was replaced with DMEM- 10% FBS supplemented with 1 mg of G418/ml.

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    NK cells release soluble factor(s) to suppress expression of <t>HCV</t> <t>RNA</t> and NS5A protein in human hepatocytes. (a) Co-culture of NK cells with HCV JFH1-infected Huh7.5.1 cells. At day 4 postinfection, Huh7.5.1 cells in the low compartment of a 24-well transwell co-culture system were co-cultured with primary NK (PNK) or NK92 cells that were added to the top compartment of the co-culture system. (b) Effect of NK SN on HCV JFH1 RNA expression. JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in media conditioned with or without NK SN (PNK or NK92) for 48 h. Total cellular RNA extracted from the hepatic cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative to the control (% of control, no NK cells on the top compartment or without treatment with NK SN, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments (*, P
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    NK cells release soluble factor(s) to suppress expression of HCV RNA and NS5A protein in human hepatocytes. (a) Co-culture of NK cells with HCV JFH1-infected Huh7.5.1 cells. At day 4 postinfection, Huh7.5.1 cells in the low compartment of a 24-well transwell co-culture system were co-cultured with primary NK (PNK) or NK92 cells that were added to the top compartment of the co-culture system. (b) Effect of NK SN on HCV JFH1 RNA expression. JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in media conditioned with or without NK SN (PNK or NK92) for 48 h. Total cellular RNA extracted from the hepatic cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative to the control (% of control, no NK cells on the top compartment or without treatment with NK SN, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments (*, P

    Journal: Journal of viral hepatitis

    Article Title: Natural killer cells suppress full cycle HCV infection of human hepatocytes

    doi: 10.1111/j.1365-2893.2008.01014.x

    Figure Lengend Snippet: NK cells release soluble factor(s) to suppress expression of HCV RNA and NS5A protein in human hepatocytes. (a) Co-culture of NK cells with HCV JFH1-infected Huh7.5.1 cells. At day 4 postinfection, Huh7.5.1 cells in the low compartment of a 24-well transwell co-culture system were co-cultured with primary NK (PNK) or NK92 cells that were added to the top compartment of the co-culture system. (b) Effect of NK SN on HCV JFH1 RNA expression. JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in media conditioned with or without NK SN (PNK or NK92) for 48 h. Total cellular RNA extracted from the hepatic cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative to the control (% of control, no NK cells on the top compartment or without treatment with NK SN, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments (*, P

    Article Snippet: To determine the amount of HCV RNA in culture SN, HCV RNA was extracted from 200 μ L of culture medium by TRI-Reagent-BD (Molecular Research Center, Inc., Cincinnati, OH, USA).

    Techniques: Expressing, Co-Culture Assay, Infection, Cell Culture, RNA Expression, Quantitative RT-PCR

    Time course of inhibition of HCV RNA expression by NK SN. Primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected hepatocyte cultures at day 4 postinfection. Intracellular (a) or extracellular (b) RNA was extracted from hepatocytes or culture supernatants (SN) at indicated time points and subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as either HCV RNA 10 7 copies/ μ g cellular RNA or 10 7 copies/mL SN, respectively. The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

    Journal: Journal of viral hepatitis

    Article Title: Natural killer cells suppress full cycle HCV infection of human hepatocytes

    doi: 10.1111/j.1365-2893.2008.01014.x

    Figure Lengend Snippet: Time course of inhibition of HCV RNA expression by NK SN. Primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected hepatocyte cultures at day 4 postinfection. Intracellular (a) or extracellular (b) RNA was extracted from hepatocytes or culture supernatants (SN) at indicated time points and subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as either HCV RNA 10 7 copies/ μ g cellular RNA or 10 7 copies/mL SN, respectively. The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

    Article Snippet: To determine the amount of HCV RNA in culture SN, HCV RNA was extracted from 200 μ L of culture medium by TRI-Reagent-BD (Molecular Research Center, Inc., Cincinnati, OH, USA).

    Techniques: Inhibition, RNA Expression, Infection, Quantitative RT-PCR

    Suppression of HCV RNA expression by NK SN under different conditions. Huh7.5.1 cells were cultured in media conditioned with or without primary NK (PNK) SN or NK92 SN for either 24 h prior to HCV infection, or simultaneously or 8 h postinfection. The cells were then washed five times to remove input HCV after 6 h incubation with HCV JFH1 and then cultured in the presence or absence of NK SN for 8 days. Intracellular (a) and extracellular (b) RNA extracted from hepatocytes or culture supernatants (SN) at day 8 postinfection was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as HCV RNA levels relative (%) to the control (untreated cell cultures, which are defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

    Journal: Journal of viral hepatitis

    Article Title: Natural killer cells suppress full cycle HCV infection of human hepatocytes

    doi: 10.1111/j.1365-2893.2008.01014.x

    Figure Lengend Snippet: Suppression of HCV RNA expression by NK SN under different conditions. Huh7.5.1 cells were cultured in media conditioned with or without primary NK (PNK) SN or NK92 SN for either 24 h prior to HCV infection, or simultaneously or 8 h postinfection. The cells were then washed five times to remove input HCV after 6 h incubation with HCV JFH1 and then cultured in the presence or absence of NK SN for 8 days. Intracellular (a) and extracellular (b) RNA extracted from hepatocytes or culture supernatants (SN) at day 8 postinfection was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification. Intracellular (a) and extracellular (b) HCV RNA expression is expressed as HCV RNA levels relative (%) to the control (untreated cell cultures, which are defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

    Article Snippet: To determine the amount of HCV RNA in culture SN, HCV RNA was extracted from 200 μ L of culture medium by TRI-Reagent-BD (Molecular Research Center, Inc., Cincinnati, OH, USA).

    Techniques: RNA Expression, Cell Culture, Infection, Incubation, Quantitative RT-PCR

    IFN- γ is involved in NK SN-mediated anti-HCV activity. (a) IFN- γ production by primary NK (PNK) cells and NK92 cells. IFN- γ levels in media from PNK cells (five donors) and NK-92 cells were determined by ELISA. (b) HCV JFH1-infected Huh7.5.1 cells were cultured in the presence or absence of NK SN and/or antibody (Ab) against IFN- γ . For the cultures using both NK SN and Ab to IFN- γ , the NK SN was preincubated with the Ab to IFN- γ for 30 min before being added to JFH1-infected hepatocyte cultures at day 4 postinfection. IFN- γ (1000 U/mL) alone was added to the cell cultures as a positive control to determine the neutralization ability of the Ab to IFN- γ . Mouse IgG 2A was used to determine the specificity of the Ab to IFN- γ . Total cellular RNA extracted from hepatocytes was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification at 48 h postexposure to NK SN. The data are expressed as HCV RNA levels relative (% of control, no Abs treatment and no NK SN added, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

    Journal: Journal of viral hepatitis

    Article Title: Natural killer cells suppress full cycle HCV infection of human hepatocytes

    doi: 10.1111/j.1365-2893.2008.01014.x

    Figure Lengend Snippet: IFN- γ is involved in NK SN-mediated anti-HCV activity. (a) IFN- γ production by primary NK (PNK) cells and NK92 cells. IFN- γ levels in media from PNK cells (five donors) and NK-92 cells were determined by ELISA. (b) HCV JFH1-infected Huh7.5.1 cells were cultured in the presence or absence of NK SN and/or antibody (Ab) against IFN- γ . For the cultures using both NK SN and Ab to IFN- γ , the NK SN was preincubated with the Ab to IFN- γ for 30 min before being added to JFH1-infected hepatocyte cultures at day 4 postinfection. IFN- γ (1000 U/mL) alone was added to the cell cultures as a positive control to determine the neutralization ability of the Ab to IFN- γ . Mouse IgG 2A was used to determine the specificity of the Ab to IFN- γ . Total cellular RNA extracted from hepatocytes was subjected to the real-time RT-PCR for HCV and GAPDH RNA quantification at 48 h postexposure to NK SN. The data are expressed as HCV RNA levels relative (% of control, no Abs treatment and no NK SN added, which is defined as 100%). The results shown are mean ± SD of triplicate cultures, representative of three experiments (*, P

    Article Snippet: To determine the amount of HCV RNA in culture SN, HCV RNA was extracted from 200 μ L of culture medium by TRI-Reagent-BD (Molecular Research Center, Inc., Cincinnati, OH, USA).

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Cell Culture, Positive Control, Neutralization, Quantitative RT-PCR

    Effect of NK SN on the expression of IFN- α/β in human hepatocytes. (a and c) IFN- α/β RNA expression. At day 4 postinfection, primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected Huh7.5.1 cells (25%, v/v). Total cellular RNA was extracted from the cell cultures at 12 h posttreatment with NK SN and then subjected to real-time RT-PCR for IFN- α/β and GAPDH RNA quantification. The data were expressed as IFN- α/β RNA levels relative (fold) to the control (without NK SN, which is defined as 1). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments. (b) IFN- α protein expression. HCV JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in the presence or absence NK (PNK and NK-92) SN (25%, v/v) for 48 h. SNs from the cell cultures were then harvested for IFN- α protein ELISA assay. The results shown are mean ± SD of triplicate cultures (*, P

    Journal: Journal of viral hepatitis

    Article Title: Natural killer cells suppress full cycle HCV infection of human hepatocytes

    doi: 10.1111/j.1365-2893.2008.01014.x

    Figure Lengend Snippet: Effect of NK SN on the expression of IFN- α/β in human hepatocytes. (a and c) IFN- α/β RNA expression. At day 4 postinfection, primary NK (PNK) SN or NK-92 SN was added to HCV JFH1-infected Huh7.5.1 cells (25%, v/v). Total cellular RNA was extracted from the cell cultures at 12 h posttreatment with NK SN and then subjected to real-time RT-PCR for IFN- α/β and GAPDH RNA quantification. The data were expressed as IFN- α/β RNA levels relative (fold) to the control (without NK SN, which is defined as 1). The results shown are mean ± SD of triplicate cultures, representative of three separate experiments. (b) IFN- α protein expression. HCV JFH1-infected Huh7.5.1 cells (day 4 postinfection) were cultured in the presence or absence NK (PNK and NK-92) SN (25%, v/v) for 48 h. SNs from the cell cultures were then harvested for IFN- α protein ELISA assay. The results shown are mean ± SD of triplicate cultures (*, P

    Article Snippet: To determine the amount of HCV RNA in culture SN, HCV RNA was extracted from 200 μ L of culture medium by TRI-Reagent-BD (Molecular Research Center, Inc., Cincinnati, OH, USA).

    Techniques: Expressing, RNA Expression, Infection, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay

    Detection of HCV proteins and RNA in Huh-7.5 cells transiently transfected with subgenomic and full-length HCV RNAs. (Top) Ninety-six hours after RNA transfection of Huh-7.5 cells, the monolayers were labeled with 35 S protein-labeling mixture and lysed, and NS3, NS4B, and NS5A were analyzed by immunoprecipitation, SDS- 10% PAGE, and autoradiography. The positions of the molecular-mass standards are given on the left, and HCV-specific proteins are indicated on the right. (Middle) Total cellular RNA was extracted 96 h posttransfection, and HCV RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA to the pol − negative control is shown (HCV RNA/pol − ). (Bottom) Ninety-six hours after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for HCV NS3, and analyzed by FACS. The percentages of cells expressing NS3 relative to an isotype-matched irrelevant IgG are displayed. Values of

    Journal: Journal of Virology

    Article Title: Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

    doi: 10.1128/JVI.77.5.3181-3190.2003

    Figure Lengend Snippet: Detection of HCV proteins and RNA in Huh-7.5 cells transiently transfected with subgenomic and full-length HCV RNAs. (Top) Ninety-six hours after RNA transfection of Huh-7.5 cells, the monolayers were labeled with 35 S protein-labeling mixture and lysed, and NS3, NS4B, and NS5A were analyzed by immunoprecipitation, SDS- 10% PAGE, and autoradiography. The positions of the molecular-mass standards are given on the left, and HCV-specific proteins are indicated on the right. (Middle) Total cellular RNA was extracted 96 h posttransfection, and HCV RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA to the pol − negative control is shown (HCV RNA/pol − ). (Bottom) Ninety-six hours after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for HCV NS3, and analyzed by FACS. The percentages of cells expressing NS3 relative to an isotype-matched irrelevant IgG are displayed. Values of

    Article Snippet: The transfected cells were plated in (i) 150-mm-diameter dishes for selection of G418-resistant colonies, (ii) 100-mm-diameter dishes for determining the efficiency of G418-resistant colony formation and fluorescence-activated cell sorting (FACS) analysis, (iii) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, or (iv) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies.

    Techniques: Transfection, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Negative Control, Staining, FACS, Expressing

    Effect(s) of alternative substitutions at position 2204 in NS5A on HCV RNA replication. Huh-7.5 cells were transfected with 1 μg of the SG-5′HE replicons carrying the indicated amino acid substitutions and 2 × 10 5 cells plated in 35-mm-diameter wells. After 24, 48, and 96 h in culture, total cellular RNA was extracted and HCV RNA levels were measured as described in Materials and Methods. The ratio of HCV RNA relative to the pol − defective subgenomic RNA (HCV RNA/pol − ) was plotted against the time posttransfection. The increase in HCV RNA above pol − is indicated above each bar. In this figure the levels of HCV RNA relative to the pol − are the highest we have achieved so far. When these RNAs were transfected into Huh-7.5 cells a second time, a similar trend in HCV RNA accumulation was observed.

    Journal: Journal of Virology

    Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

    doi: 10.1128/JVI.76.24.13001-13014.2002

    Figure Lengend Snippet: Effect(s) of alternative substitutions at position 2204 in NS5A on HCV RNA replication. Huh-7.5 cells were transfected with 1 μg of the SG-5′HE replicons carrying the indicated amino acid substitutions and 2 × 10 5 cells plated in 35-mm-diameter wells. After 24, 48, and 96 h in culture, total cellular RNA was extracted and HCV RNA levels were measured as described in Materials and Methods. The ratio of HCV RNA relative to the pol − defective subgenomic RNA (HCV RNA/pol − ) was plotted against the time posttransfection. The increase in HCV RNA above pol − is indicated above each bar. In this figure the levels of HCV RNA relative to the pol − are the highest we have achieved so far. When these RNAs were transfected into Huh-7.5 cells a second time, a similar trend in HCV RNA accumulation was observed.

    Article Snippet: Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection.

    Techniques: Transfection

    Effect(s) of S2194A and S2194D mutations on HCV RNA replication. S2194 was replaced with Ala or Asp in the selectable bicistronic replicon SG-Neo (S2204I), and RNA was transcribed in vitro. (A) RNA transcripts were transfected into Huh-7 cells, and G418-selected colonies were fixed and stained with crystal violet. The relative G418 transduction efficiencies are indicated below each dish. (B) Ninety-six hours posttransfection Huh-7.5 cells were labeled with [ 35 S]methionine and [ 35 S]cysteine for 10 h. Cells were lysed, and HCV proteins were isolated by immunoprecipitation using a patient serum specific for NS3, NS4B, and NS5A. HCV proteins and the positions of protein molecular weight standards (in thousands) are shown. The ratio of HCV RNA relative to the pol − negative control at 96 h posttransfection is shown below each track (HCV RNA/pol − ). The results illustrated are representative of two independent transfections.

    Journal: Journal of Virology

    Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

    doi: 10.1128/JVI.76.24.13001-13014.2002

    Figure Lengend Snippet: Effect(s) of S2194A and S2194D mutations on HCV RNA replication. S2194 was replaced with Ala or Asp in the selectable bicistronic replicon SG-Neo (S2204I), and RNA was transcribed in vitro. (A) RNA transcripts were transfected into Huh-7 cells, and G418-selected colonies were fixed and stained with crystal violet. The relative G418 transduction efficiencies are indicated below each dish. (B) Ninety-six hours posttransfection Huh-7.5 cells were labeled with [ 35 S]methionine and [ 35 S]cysteine for 10 h. Cells were lysed, and HCV proteins were isolated by immunoprecipitation using a patient serum specific for NS3, NS4B, and NS5A. HCV proteins and the positions of protein molecular weight standards (in thousands) are shown. The ratio of HCV RNA relative to the pol − negative control at 96 h posttransfection is shown below each track (HCV RNA/pol − ). The results illustrated are representative of two independent transfections.

    Article Snippet: Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection.

    Techniques: In Vitro, Transfection, Staining, Transduction, Labeling, Isolation, Immunoprecipitation, Molecular Weight, Negative Control

    Detection of HCV proteins and RNA in Huh-7.5 and Huh-7 cells transiently transfected with HCV RNA. Top panel, Huh-7.5 and Huh-7 cells were transfected with the subgenomic replicons pol − (lanes 1 and 7), SG-5′HE (S2204I) (lanes 2 and 8), SG-5′HE (5AΔ47) (lanes 3 and 9), SG-Neo (S2204I) (lanes 4 and 10), SG-Neo (5AΔ47) (lanes 5 and 11), and FL (S2204I) HCV RNA (lanes 6 and 12). At 96 h posttransfection, monolayers were incubated for 10 h in the presence of [ 35 S]methionine and [ 35 S]cysteine. Labeled cells were lysed and immunoprecipitated with HCV-positive human serum (anti-NS3, NS4B, and NS5A), and labeled proteins were separated by SDS-10% PAGE. Note that twice the amount of immunoprecipitated sample was loaded in lanes 6 and 12. The mobilities of molecular weight standards (in thousands) are indicated on the left, and the migration of NS3, NS4B, NS5A, and 5AΔ47 is shown on the right. For data in the panel directly below the gel, total cellular RNA was extracted at 96 h posttransfection and quantified for HCV RNA levels as described in the Materials and Methods. The ratio of HCV RNA relative to the pol − defective replicon is shown (HCV RNA/pol − ). HCV RNA levels relative to the pol − control were comparable in three independent experiments. For data in the bottom two panels, 96 h after transfection cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for either HCV core or NS3 antigens, and analyzed by FACS. The percentage of cells expressing core and NS3 relative to an isotype matched irrelevant IgG is displayed. Values

    Journal: Journal of Virology

    Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

    doi: 10.1128/JVI.76.24.13001-13014.2002

    Figure Lengend Snippet: Detection of HCV proteins and RNA in Huh-7.5 and Huh-7 cells transiently transfected with HCV RNA. Top panel, Huh-7.5 and Huh-7 cells were transfected with the subgenomic replicons pol − (lanes 1 and 7), SG-5′HE (S2204I) (lanes 2 and 8), SG-5′HE (5AΔ47) (lanes 3 and 9), SG-Neo (S2204I) (lanes 4 and 10), SG-Neo (5AΔ47) (lanes 5 and 11), and FL (S2204I) HCV RNA (lanes 6 and 12). At 96 h posttransfection, monolayers were incubated for 10 h in the presence of [ 35 S]methionine and [ 35 S]cysteine. Labeled cells were lysed and immunoprecipitated with HCV-positive human serum (anti-NS3, NS4B, and NS5A), and labeled proteins were separated by SDS-10% PAGE. Note that twice the amount of immunoprecipitated sample was loaded in lanes 6 and 12. The mobilities of molecular weight standards (in thousands) are indicated on the left, and the migration of NS3, NS4B, NS5A, and 5AΔ47 is shown on the right. For data in the panel directly below the gel, total cellular RNA was extracted at 96 h posttransfection and quantified for HCV RNA levels as described in the Materials and Methods. The ratio of HCV RNA relative to the pol − defective replicon is shown (HCV RNA/pol − ). HCV RNA levels relative to the pol − control were comparable in three independent experiments. For data in the bottom two panels, 96 h after transfection cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for either HCV core or NS3 antigens, and analyzed by FACS. The percentage of cells expressing core and NS3 relative to an isotype matched irrelevant IgG is displayed. Values

    Article Snippet: Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection.

    Techniques: Transfection, Incubation, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Molecular Weight, Migration, Staining, FACS, Expressing

    HCV RNA accumulation after transfection of Huh-7.5 cells with full-length HCV RNA. One microgram of in vitro-transcribed RNA was electroporated into Huh-7.5, and 2 × 10 5 cells were plated into 35-mm-diameter wells. Total cellular RNA was isolated at 24, 48, and 96 h posttransfection, and HCV RNA levels were quantified as described in the Materials and Methods. The ratio of HCV RNA relative to the pol − defective subgenomic RNA (HCV RNA/pol − ) was plotted against the time posttransfection, and similar results were obtained when this experiment was repeated

    Journal: Journal of Virology

    Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

    doi: 10.1128/JVI.76.24.13001-13014.2002

    Figure Lengend Snippet: HCV RNA accumulation after transfection of Huh-7.5 cells with full-length HCV RNA. One microgram of in vitro-transcribed RNA was electroporated into Huh-7.5, and 2 × 10 5 cells were plated into 35-mm-diameter wells. Total cellular RNA was isolated at 24, 48, and 96 h posttransfection, and HCV RNA levels were quantified as described in the Materials and Methods. The ratio of HCV RNA relative to the pol − defective subgenomic RNA (HCV RNA/pol − ) was plotted against the time posttransfection, and similar results were obtained when this experiment was repeated

    Article Snippet: Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection.

    Techniques: Transfection, In Vitro, Isolation

    Effect(s) of combining NS3 and NS5A mutations on HCV RNA replication. Subgenomic replicons lacking neo (SG-5′HE) were generated carrying S2204I with further mutations in NS3. (A) For the gel shown at top, 96 h after RNA transfection of Huh-7.5 cells, monolayers were labeled with 35 S-protein labeling mixture; cells were lysed; and NS3, NS4A, and NS5A were analyzed by immunoprecipitation, SDS-10% PAGE, and autoradiography. Positions of the molecular weight standards (in thousands) are given on the left, and HCV-specific proteins are indicated to the right. For data in the panel directly below the gel, total cellular RNA was extracted at 96 h posttransfection and HCV RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA relative to the pol − negative control is shown (HCV RNA/pol − ). Comparable ratios were obtained in two independent experiments. For data in the bottom two panels, 96 h after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for HCV NS3 and NS5B antigens, and analyzed by FACS. The percentage of cells expressing NS3 and NS5B relative to an isotype-matched irrelevant IgG is displayed. Values

    Journal: Journal of Virology

    Article Title: Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

    doi: 10.1128/JVI.76.24.13001-13014.2002

    Figure Lengend Snippet: Effect(s) of combining NS3 and NS5A mutations on HCV RNA replication. Subgenomic replicons lacking neo (SG-5′HE) were generated carrying S2204I with further mutations in NS3. (A) For the gel shown at top, 96 h after RNA transfection of Huh-7.5 cells, monolayers were labeled with 35 S-protein labeling mixture; cells were lysed; and NS3, NS4A, and NS5A were analyzed by immunoprecipitation, SDS-10% PAGE, and autoradiography. Positions of the molecular weight standards (in thousands) are given on the left, and HCV-specific proteins are indicated to the right. For data in the panel directly below the gel, total cellular RNA was extracted at 96 h posttransfection and HCV RNA levels were quantified as described in Materials and Methods. The ratio of HCV RNA relative to the pol − negative control is shown (HCV RNA/pol − ). Comparable ratios were obtained in two independent experiments. For data in the bottom two panels, 96 h after transfection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, stained for HCV NS3 and NS5B antigens, and analyzed by FACS. The percentage of cells expressing NS3 and NS5B relative to an isotype-matched irrelevant IgG is displayed. Values

    Article Snippet: Cells were plated in (i) 35-mm-diameter wells for quantifying HCV RNA and for metabolic labeling experiments, (ii) eight-well chamber slides (Becton Dickinson) for immunofluorescence studies, or (iii) 100-mm-diameter dishes for fluorescence-activated cell sorting (FACS) analysis and G418 selection.

    Techniques: Generated, Transfection, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography, Molecular Weight, Negative Control, Staining, FACS, Expressing