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A Dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus.

Ren Q, Li C, Yuan P, Cai C, Zhang L, Luo GG, Wei W - Sci Rep (2015)

Bottom Line: The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide.Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV.The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

View Article: PubMed Central - PubMed

Affiliation: Biodynamic Optical Imaging Center (BIOPIC), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.

ABSTRACT
The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide. Although several reporter systems have been developed, many shortcomings limit their use in the assessment of HCV infections. Here, we report a real-time live-cell reporter, termed the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, which provides an on-off switch specifically in response to an HCV infection. Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV. The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

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Quantitative evaluation of NIrD system in response to HCV inoculation.(a) Time-lapse live-cell imaging of Huh7.5(NIrD) cells. Both light and fluorescence images were taken every 24 h, starting at 24 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (b) FACS analysis of Huh7.5(NIrD) cells infected by HCVcc in the presence of Dox (2 μg/ml). A total of 2 × 105/well of Huh7.5(NIrD) cells were seeded in 6-well plates. Representative results from reporter cells treated with HCVcc (0 or 25,100 TCID50/ml) are presented. FACS analysis was conducted 96 h following the viral infection. The numbers in the square indicate the percentage of red fluorescence-negative cells. (c-d) FACS analysis of Huh7.5(NIrD) cells infected by serially increasing dosages of HCVcc. The curves show the percentage of mCherry positive cells corresponding to MOI (2 × 105 cells/well) in linear (c) or logarithmic (d) plots.
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f2: Quantitative evaluation of NIrD system in response to HCV inoculation.(a) Time-lapse live-cell imaging of Huh7.5(NIrD) cells. Both light and fluorescence images were taken every 24 h, starting at 24 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (b) FACS analysis of Huh7.5(NIrD) cells infected by HCVcc in the presence of Dox (2 μg/ml). A total of 2 × 105/well of Huh7.5(NIrD) cells were seeded in 6-well plates. Representative results from reporter cells treated with HCVcc (0 or 25,100 TCID50/ml) are presented. FACS analysis was conducted 96 h following the viral infection. The numbers in the square indicate the percentage of red fluorescence-negative cells. (c-d) FACS analysis of Huh7.5(NIrD) cells infected by serially increasing dosages of HCVcc. The curves show the percentage of mCherry positive cells corresponding to MOI (2 × 105 cells/well) in linear (c) or logarithmic (d) plots.

Mentions: To quantitatively evaluate the NIrD system in response to HCV infections, we monitored the mCherry signal upon viral inoculation at different time points. The red fluorescence did not appear until 48 h after the infection, and the number of cells showing fluorescence increased over time (Fig. 2a). Uninfected cells showed no detectable signals in FACS analysis, while Huh7.5(NIrD) responded with high sensitivity to the viral infection; in total, 96.6% of infected Huh7.5(NIrD) cells displayed red fluorescence when the titre of HCVcc reached 2.51 × 104 TCID50/ml (Fig. 2b). These results indicate a superior signal/noise ratio, a finding that was further confirmed by detailed FACS analysis, which showed that the reporter signals and HCV titres had a strong linear correlation (R2 = 0.995) within a dynamic range (Fig. 2c-d). In particular, the NIrD system was sensitive enough to detect HCV with a MOI as low as 0.001 (Fig. 2d).


A Dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus.

Ren Q, Li C, Yuan P, Cai C, Zhang L, Luo GG, Wei W - Sci Rep (2015)

Quantitative evaluation of NIrD system in response to HCV inoculation.(a) Time-lapse live-cell imaging of Huh7.5(NIrD) cells. Both light and fluorescence images were taken every 24 h, starting at 24 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (b) FACS analysis of Huh7.5(NIrD) cells infected by HCVcc in the presence of Dox (2 μg/ml). A total of 2 × 105/well of Huh7.5(NIrD) cells were seeded in 6-well plates. Representative results from reporter cells treated with HCVcc (0 or 25,100 TCID50/ml) are presented. FACS analysis was conducted 96 h following the viral infection. The numbers in the square indicate the percentage of red fluorescence-negative cells. (c-d) FACS analysis of Huh7.5(NIrD) cells infected by serially increasing dosages of HCVcc. The curves show the percentage of mCherry positive cells corresponding to MOI (2 × 105 cells/well) in linear (c) or logarithmic (d) plots.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352851&req=5

f2: Quantitative evaluation of NIrD system in response to HCV inoculation.(a) Time-lapse live-cell imaging of Huh7.5(NIrD) cells. Both light and fluorescence images were taken every 24 h, starting at 24 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (b) FACS analysis of Huh7.5(NIrD) cells infected by HCVcc in the presence of Dox (2 μg/ml). A total of 2 × 105/well of Huh7.5(NIrD) cells were seeded in 6-well plates. Representative results from reporter cells treated with HCVcc (0 or 25,100 TCID50/ml) are presented. FACS analysis was conducted 96 h following the viral infection. The numbers in the square indicate the percentage of red fluorescence-negative cells. (c-d) FACS analysis of Huh7.5(NIrD) cells infected by serially increasing dosages of HCVcc. The curves show the percentage of mCherry positive cells corresponding to MOI (2 × 105 cells/well) in linear (c) or logarithmic (d) plots.
Mentions: To quantitatively evaluate the NIrD system in response to HCV infections, we monitored the mCherry signal upon viral inoculation at different time points. The red fluorescence did not appear until 48 h after the infection, and the number of cells showing fluorescence increased over time (Fig. 2a). Uninfected cells showed no detectable signals in FACS analysis, while Huh7.5(NIrD) responded with high sensitivity to the viral infection; in total, 96.6% of infected Huh7.5(NIrD) cells displayed red fluorescence when the titre of HCVcc reached 2.51 × 104 TCID50/ml (Fig. 2b). These results indicate a superior signal/noise ratio, a finding that was further confirmed by detailed FACS analysis, which showed that the reporter signals and HCV titres had a strong linear correlation (R2 = 0.995) within a dynamic range (Fig. 2c-d). In particular, the NIrD system was sensitive enough to detect HCV with a MOI as low as 0.001 (Fig. 2d).

Bottom Line: The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide.Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV.The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

View Article: PubMed Central - PubMed

Affiliation: Biodynamic Optical Imaging Center (BIOPIC), Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China.

ABSTRACT
The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide. Although several reporter systems have been developed, many shortcomings limit their use in the assessment of HCV infections. Here, we report a real-time live-cell reporter, termed the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, which provides an on-off switch specifically in response to an HCV infection. Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV. The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

Show MeSH
Related in: MedlinePlus