Limits...
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.

Show MeSH

Related in: MedlinePlus

A cell-based dual-reporter system for monitoring HCV infections.(a) The rationale of the NIrD system. The sensor module consists of rtTA-MAVS(C) fusion proteins, which are constantly produced and localised to the mitochondria in cells. The amplifier module is an expression cartridge integrated into the chromosome that is composed of two reporter genes driven by the tight-TRE promoter. Upon HCV infection, the virally produced NS3-4A cleaves its recognition sequence in MAVS(C), releasing the free-formed rtTA into the nucleus. The tight-TRE promoter is activated by rtTA and Dox (2 μg/ml), resulting in the production of delta-TK-2A-mCherry proteins. After self-cleavage of the 2A peptide, mCherry shows red fluorescence and delta-TK phosphorylates GCV (2 μg/ml) to cause cell death. (b) mCherry signal of the NIrD system in response to an HCVcc infection. The Huh7.5(NIrD) system was infected by HCVcc for 72 h. HCVcc-transduced (+) or -untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a microscope. The fluorescence signals were superimposed onto white light images. Scale bar, 200 μm. (c) The death signal of the NIrD system in response to an HCVcc infection in the presence of GCV (2 μg/ml). Huh7.5(NIrD) cells were infected with HCVcc for 120 h. The transduced (+) or untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a light microscope. Scale bar, 200 μm. (d) Fluorescence microscopy of HCVcc core protein (Alexa Fluor 488) and mCherry signal upon HCVcc infection (72 h) in Huh7.5(NIrD) cells. Scale bar, 30 μm. (e) Immunoblotting analysis of Huh7.5(NIrD) cells infected with or without HCVcc in the presence or absence of Dox (2 μg/ml). HCVcc was detected by an antibody specifically targeting the viral core protein, and β-tubulin was used as the loading control. (f) Dosage effects of VX-950 on the live-cell imaging of HCVcc-infected Huh7.5(NIrD) cells. Huh7.5(NIrD) cells were infected with HCVcc plus Dox (2 μg/ml) together with serially increasing dosages of VX-950 (Telaprevir). Fluorescence microscopic images were taken 72 h following the HCVcc infection. Scale bar, 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4352851&req=5

f1: A cell-based dual-reporter system for monitoring HCV infections.(a) The rationale of the NIrD system. The sensor module consists of rtTA-MAVS(C) fusion proteins, which are constantly produced and localised to the mitochondria in cells. The amplifier module is an expression cartridge integrated into the chromosome that is composed of two reporter genes driven by the tight-TRE promoter. Upon HCV infection, the virally produced NS3-4A cleaves its recognition sequence in MAVS(C), releasing the free-formed rtTA into the nucleus. The tight-TRE promoter is activated by rtTA and Dox (2 μg/ml), resulting in the production of delta-TK-2A-mCherry proteins. After self-cleavage of the 2A peptide, mCherry shows red fluorescence and delta-TK phosphorylates GCV (2 μg/ml) to cause cell death. (b) mCherry signal of the NIrD system in response to an HCVcc infection. The Huh7.5(NIrD) system was infected by HCVcc for 72 h. HCVcc-transduced (+) or -untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a microscope. The fluorescence signals were superimposed onto white light images. Scale bar, 200 μm. (c) The death signal of the NIrD system in response to an HCVcc infection in the presence of GCV (2 μg/ml). Huh7.5(NIrD) cells were infected with HCVcc for 120 h. The transduced (+) or untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a light microscope. Scale bar, 200 μm. (d) Fluorescence microscopy of HCVcc core protein (Alexa Fluor 488) and mCherry signal upon HCVcc infection (72 h) in Huh7.5(NIrD) cells. Scale bar, 30 μm. (e) Immunoblotting analysis of Huh7.5(NIrD) cells infected with or without HCVcc in the presence or absence of Dox (2 μg/ml). HCVcc was detected by an antibody specifically targeting the viral core protein, and β-tubulin was used as the loading control. (f) Dosage effects of VX-950 on the live-cell imaging of HCVcc-infected Huh7.5(NIrD) cells. Huh7.5(NIrD) cells were infected with HCVcc plus Dox (2 μg/ml) together with serially increasing dosages of VX-950 (Telaprevir). Fluorescence microscopic images were taken 72 h following the HCVcc infection. Scale bar, 200 μm.

Mentions: The design of this novel HCV reporter consists of two modules, a sensor and an amplifier. The sensor is the chimeric protein rtTA-MAVS(C) (reverse tetracycline transactivator2324 - mitochondrial antiviral signalling protein25 (C-terminal amino acids 462–540)) that contains the NS3-4A cleavage site. The amplifier is an expression module composed of the tight-TRE promoter26 followed by the coding sequences of 2A-linked delta-TK27 and mCherry (Fig. 1a). To minimise a potential leakage problem, the sensor and amplifier are spatially separated, with the former anchored to the cytoplasmic mitochondria and the latter located in the nucleus. In addition, the activation of the tight-TRE promoter requires both rtTA and doxycycline (Dox)/tetracycline, further minimising its non-specific activation. This live cell reporter, designated as the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, provides an on-off switch that specifically responds to an HCV invasion. Upon inoculation, HCV-encoded NS3-4A protease cleaves rtTA-MAVS(C) in mitochondria, and the free-formed rtTA subsequently enters the nucleus, where it binds to and activates the tight-TRE promoter in the presence of Dox, resulting in de novo expression of delta-TK-2A-mCherry. After 2A-mediated cleavage, mCherry gives rise to red fluorescence and delta-TK leads to cell death in the presence of GCV (Ganciclovir)27 (Fig. 1a). All elements of the sensor and amplifier were combined into a single lentiviral backbone, pLenti-NIrD (Supplementary Fig. 1), making it convenient to acquire stable clones with the integrated NIrD system in any given cell type through viral infection and Blasticidin selection.


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)

A cell-based dual-reporter system for monitoring HCV infections.(a) The rationale of the NIrD system. The sensor module consists of rtTA-MAVS(C) fusion proteins, which are constantly produced and localised to the mitochondria in cells. The amplifier module is an expression cartridge integrated into the chromosome that is composed of two reporter genes driven by the tight-TRE promoter. Upon HCV infection, the virally produced NS3-4A cleaves its recognition sequence in MAVS(C), releasing the free-formed rtTA into the nucleus. The tight-TRE promoter is activated by rtTA and Dox (2 μg/ml), resulting in the production of delta-TK-2A-mCherry proteins. After self-cleavage of the 2A peptide, mCherry shows red fluorescence and delta-TK phosphorylates GCV (2 μg/ml) to cause cell death. (b) mCherry signal of the NIrD system in response to an HCVcc infection. The Huh7.5(NIrD) system was infected by HCVcc for 72 h. HCVcc-transduced (+) or -untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a microscope. The fluorescence signals were superimposed onto white light images. Scale bar, 200 μm. (c) The death signal of the NIrD system in response to an HCVcc infection in the presence of GCV (2 μg/ml). Huh7.5(NIrD) cells were infected with HCVcc for 120 h. The transduced (+) or untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a light microscope. Scale bar, 200 μm. (d) Fluorescence microscopy of HCVcc core protein (Alexa Fluor 488) and mCherry signal upon HCVcc infection (72 h) in Huh7.5(NIrD) cells. Scale bar, 30 μm. (e) Immunoblotting analysis of Huh7.5(NIrD) cells infected with or without HCVcc in the presence or absence of Dox (2 μg/ml). HCVcc was detected by an antibody specifically targeting the viral core protein, and β-tubulin was used as the loading control. (f) Dosage effects of VX-950 on the live-cell imaging of HCVcc-infected Huh7.5(NIrD) cells. Huh7.5(NIrD) cells were infected with HCVcc plus Dox (2 μg/ml) together with serially increasing dosages of VX-950 (Telaprevir). Fluorescence microscopic images were taken 72 h following the HCVcc infection. Scale bar, 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: A cell-based dual-reporter system for monitoring HCV infections.(a) The rationale of the NIrD system. The sensor module consists of rtTA-MAVS(C) fusion proteins, which are constantly produced and localised to the mitochondria in cells. The amplifier module is an expression cartridge integrated into the chromosome that is composed of two reporter genes driven by the tight-TRE promoter. Upon HCV infection, the virally produced NS3-4A cleaves its recognition sequence in MAVS(C), releasing the free-formed rtTA into the nucleus. The tight-TRE promoter is activated by rtTA and Dox (2 μg/ml), resulting in the production of delta-TK-2A-mCherry proteins. After self-cleavage of the 2A peptide, mCherry shows red fluorescence and delta-TK phosphorylates GCV (2 μg/ml) to cause cell death. (b) mCherry signal of the NIrD system in response to an HCVcc infection. The Huh7.5(NIrD) system was infected by HCVcc for 72 h. HCVcc-transduced (+) or -untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a microscope. The fluorescence signals were superimposed onto white light images. Scale bar, 200 μm. (c) The death signal of the NIrD system in response to an HCVcc infection in the presence of GCV (2 μg/ml). Huh7.5(NIrD) cells were infected with HCVcc for 120 h. The transduced (+) or untransduced (−) cells in the presence (+) or absence (−) of Dox (2 μg/ml) were visualised under a light microscope. Scale bar, 200 μm. (d) Fluorescence microscopy of HCVcc core protein (Alexa Fluor 488) and mCherry signal upon HCVcc infection (72 h) in Huh7.5(NIrD) cells. Scale bar, 30 μm. (e) Immunoblotting analysis of Huh7.5(NIrD) cells infected with or without HCVcc in the presence or absence of Dox (2 μg/ml). HCVcc was detected by an antibody specifically targeting the viral core protein, and β-tubulin was used as the loading control. (f) Dosage effects of VX-950 on the live-cell imaging of HCVcc-infected Huh7.5(NIrD) cells. Huh7.5(NIrD) cells were infected with HCVcc plus Dox (2 μg/ml) together with serially increasing dosages of VX-950 (Telaprevir). Fluorescence microscopic images were taken 72 h following the HCVcc infection. Scale bar, 200 μm.
Mentions: The design of this novel HCV reporter consists of two modules, a sensor and an amplifier. The sensor is the chimeric protein rtTA-MAVS(C) (reverse tetracycline transactivator2324 - mitochondrial antiviral signalling protein25 (C-terminal amino acids 462–540)) that contains the NS3-4A cleavage site. The amplifier is an expression module composed of the tight-TRE promoter26 followed by the coding sequences of 2A-linked delta-TK27 and mCherry (Fig. 1a). To minimise a potential leakage problem, the sensor and amplifier are spatially separated, with the former anchored to the cytoplasmic mitochondria and the latter located in the nucleus. In addition, the activation of the tight-TRE promoter requires both rtTA and doxycycline (Dox)/tetracycline, further minimising its non-specific activation. This live cell reporter, designated as the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, provides an on-off switch that specifically responds to an HCV invasion. Upon inoculation, HCV-encoded NS3-4A protease cleaves rtTA-MAVS(C) in mitochondria, and the free-formed rtTA subsequently enters the nucleus, where it binds to and activates the tight-TRE promoter in the presence of Dox, resulting in de novo expression of delta-TK-2A-mCherry. After 2A-mediated cleavage, mCherry gives rise to red fluorescence and delta-TK leads to cell death in the presence of GCV (Ganciclovir)27 (Fig. 1a). All elements of the sensor and amplifier were combined into a single lentiviral backbone, pLenti-NIrD (Supplementary Fig. 1), making it convenient to acquire stable clones with the integrated NIrD system in any given cell type through viral infection and Blasticidin selection.

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