Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α

Figures

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  Figure 1. Construction of SARS-CoV-2 subreplicon RNA expression vector. (A) Schematic structure of SARS-CoV-2 genomic RNA and SARS-CoV-2 subreplicon RNA. SARS-CoV-2 subreplicon RNA expression vector contains 5′-UTR (1–265), a part of ORF1a (266–475, green box), nucleocapsid (N, 28277–29530, blue box), and ORF10 (open box) and 3′-UTR (29531–29870), and the firefly luciferase sequence was inserted between ORF1a and nucleocapsid. The secondary structure of SARS-CoV-2 genomic RNA presented by Wacker et al. is schematically shown [23], and the protein translated regions of ORF1a, luciferase, and nucleocapsid are boxed and colored with green, yellow, and blue, respectively. Spike (S), membrane (M), and nucleocapsid (N) regions are boxed and marked S, M, and N, respectively. SARS-CoV-2 subreplicon RNA is numbered based on the Gene Bank MN994467 SARS-CoV-2 genomic RNA sequence. (B) SARS-CoV-2 subreplicon cDNA was inserted into the CMV promoter (orange arrow) for construction of the expression vector. Lengths of SARS-CoV-2 subreplicon cDNA regions are numbered. SARS-CoV-2 subreplicon RNA is expressed with poly-A tail (purple box). A fusion protein of part of ORF1a, firefly luciferase, and…

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  Figure 2. Measurement of IFN-α and CDM-3008 anti-SARS-CoV-2 activity using SARS-CoV-2 subreplicon RNA-expressing Calu-3 cells. (A) Schematic of the experimental design of transfection with SARS-CoV-2 subreplicon RNA expression vector and treatments of IFN-α and CDM-3008 for 3 days prior to luciferase and cell viability assays. (B,C) SARS-CoV-2 subreplicon RNA-expressing Calu-3 cells were treated with 1–10,000 IU/mL IFN-α in B and 0.001-10 μM CDM-3008 in C for 1 day. Luciferase intensity (blue bars) and cell viability (orange lines) were measured and are shown as % of DMSO control. Error bars indicate SD (n = 3). * p < 0.05 and ** p < 0.01 (two-tailed t-test).

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  Figure 3. Measurement of IFN-α and CDM-3008 anti-SARS-CoV-2 activity using SARS-CoV-2 subreplicon expression vector-electroporated HeLa cells. (A) Schematic of the experimental design of electroporation with SARS-CoV-2 subreplicon expression vector and treatments of IFN-α and CDM-3008 for 1 day prior to luciferase and cell viability assays. (B,C) SARS-CoV-2 subreplicon-expressing HeLa cells were treated with 1–10,000 IU/mL IFN-α in B and 0.01-100 μM CDM-3008 in C for 1 day. Luciferase intensity (blue bars) and cell viability (orange lines) were measured and are shown as % of DMSO control. Error bars indicate SD (n = 3). * p < 0.05 and ** p < 0.01 (two-tailed t-test).

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  Figure 4. Measurement of IFN-α anti-SARS-CoV-2 activity using HeLa cells stably expressing SARS-CoV-2 subreplicon. (A) Schematic of the experimental design of treatments of HeLa cells stably expressing SARS-CoV-2 subreplicon (3C5) with IFN-α and Pyridone 6 for 1 day prior to luciferase and cell viability assays. (B,C) HeLa cells stably expressing SARS-CoV-2 subreplicon were treated with 100–100,000 IU/mL IFN-α in B and simultaneously treated with 10 μM Pyridone 6 in C for 1 day. Luciferase intensity (blue bars) and cell viability (orange lines) were measured and are shown as % of DMSO control. Error bars indicate SD (n = 3). * p < 0.05 and ** p < 0.01 (two-tailed t-test).

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  Figure 5. Determination of ISGs suppressing SARS-CoV-2 genomic RNA using siRNA. (A) Schematic of the experimental design of HeLa cells stably expressing SARS-CoV-2 subreplicon (3C5) treated with siRNA and IFN-α for 1 day each prior to luciferase and cell viability assays. (B) HeLa cells stably expressing SARS-CoV-2 subreplicon were treated with OAS1, 2, and 3 and ISG20 siRNA for 1 day, and then the cells were treated with 1000 IU/mL IFN-α for 1 day. Luciferase intensity (blue bars) and cell viability (orange line) were measured and are shown as % of control siRNA-treated DMSO control. Error bars indicate SD (n = 3). * p < 0.05 (two-tailed t-test).