A Dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus - PubMed (original) (raw)

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

Qingpeng Ren et al. Sci Rep. 2015.

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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Figures

Figure 1

Figure 1. 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.

Figure 2

Figure 2. 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.

Figure 3

Figure 3. Schematic of the CRISPR library construction and HCV screening.

(a) The structure of the lentiviral plasmid expressing OCT1 and Cas9. (b) Indels induced by the lentivirus-delivered sgRNA (5-TTGGCCAGACTTGCATCCG-3) targeting the CSPG4 gene in the indicated cells were assayed by T7E1 digestion. Genomic DNA from HeLaOC-SC was used as a positive control, and the wild type (WT) Huh7.5(NIrD) was used as a negative control. (c) Schematic of the sgRNA library screening. sgRNAs were delivered into Huh7.5(NIrD)OC-SC cells by lentiviral infection with a MOI of 0.1. Three replicates of the libraries were challenged with 3–4 rounds of HCVcc, followed by FACS sorting to enrich the mCherry-negative clones. A comparison of the abundance of sgRNAs between the treated and untreated populations through high-throughput sequencing analysis was conducted following the same procedure as previously reported.

Figure 4

Figure 4. Screen of host genes essential for an HCV infection.

(a) Primary HCVcc screening data. The count of every sgRNA is the number of reads that match the sgRNA target sequence. (b) sgRNA ranking was based on the fold change of normalised counts of every sgRNA in the HCV treated and untreated populations. (c) The FDR (false discovery rate) of every gene in the library was calculated by MAGeCK based on the counts and kinds of sgRNAs in the three replicates. (d) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-free entry of HCVcc. All cells indicated carry the NIrD system. Light and fluorescence images were taken 72 h post-HCVcc infection in the presence of Dox (2 μg/ml). Scale bar, 100 μm. (e) Effects of the gene knockout of CLND1, OCLN and CD81 on cell-to-cell transmission of HCVcc. Huh7.5(NΙrD) cells with the indicated background (WT, _CLDN1_−/−, _CLDN1_−/−/CLDN1, _OCLN_−/−, _OCLN_−/−/OCLN, _CD81_−/− or _CD81_−/−/CD81) were co-cultured with HCVcc pre-infected (24 h prior) Huh7.5 cells. HCVcc carries the EGFP gene in its genome, resulting in a punctuated green fluorescence pattern in the cells. _OCLN_−/− and _CD81_−/− knockout cells expressed diffused green fluorescence because they were derived from cells expressing EGFP. The light and fluorescence (green and red) images were taken 72 h following the co-culturing of the cells. Scale bar, 100 μm.

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