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. 2021 Dec 10;12:7193. doi: 10.1038/s41467-021-27431-0

Fig. 2. A functional screen of SARS-CoV-2 –1PRF element interactors.

Fig. 2

A Schematic representation of the dual-fluorescence frameshift reporter construct. EGFP and mCherry are separated by a self-cleaving 2 A peptide as well as by a stop codon in-frame with EGFP. As a result, 0-frame translation would produce only EGFP, whereas –1PRF would produce both EGFP and mCherry. The ratio of mCherry to EGFP fluorescence is used to quantify the FE. The trans-factor construct is an N-terminal fusion of ECFP with the protein of interest to be analyzed. The control construct consists of ECFP alone. B Confocal microscopy images of cells transfected with the EGFP-mCherry control (CC- no –1PRF site included after EGFP and mCherry in-frame with EGFP), –1PRF, and no PRF (no –1PRF site and stop codon after EGFP) constructs. The size bar represents 50 µm. n = 1 independent experiment. C Comparison of relative FE of cells overexpressing trans-factors as ECFP fusion proteins. Data points represent the mean ± s.d. (n = 3 independent experiments). P values were calculated using an ordinary unpaired one-sided ANOVA comparing every condition to the ECFP control. ZAP-L and ZAP-S were separately compared to each other. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values: SFL – 0.03, DDX3 – 0.99, DDX17 – 0.72, ELAVL1 – < 0.0001, GNL2 – 0.99, GRSF – 0.03, HNRNPF – 0.99, HNRNPH1 – < 0.0001, HNRNPH2 – < 0.0001, IMP1 – 0.39, IMP3 – 0.68, PAPD4 – 0.01, PINX – 0.74, POP1 – 0.0005, SART – 0.36, SSB – 0.99, ZAP-L – 0.001, ZAP-S – < 0.0001, ZFR – 0.12. D Virus titers in the supernatant of infected naïve Huh7 or ZAP-S overexpressing Huh7 cells (ZAP-S OE) at 24 h post infection. Treatment with IFN-ɣ (500 U/ml), IFN-β (500 U/ml), or IFN-ƛ1 (5 ng/ml) was done 1 h before infection. Boxes show mean values ± s.d. (n = 4 independent experiments). The dotted line represents the limit of detection (LOD). P values were calculated using an ordinary unpaired one-sided ANOVA comparing every condition to untreated naïve infected Huh7 cells. Exact P values: untreated +ZAP-S – 0.01, INF-α2 + ZAP-S – 0.04, INF-ß + ZAP-S – 0.49, INF-γ + ZAP-S – 0.049, INF-λ + ZAP-S – 0.049. E In vivo dual-fluorescence of additional –1PRF RNAs in HEK293 cells in the presence and absence of ZAP-S. SARS-CoV-1 – severe acute respiratory syndrome-related coronavirus 1, MERS-CoV – Middle East respiratory syndrome-related coronavirus, Bat-CoV-273 – Bat Coronavirus 273, HKU1 – Human coronavirus HKU1, OC43 – Human Coronavirus OC43, CHIKV – Chikungunya Virus, HIV-1 – Human Immunodeficiency Virus 1, JEV – Japanese Encephalitis Virus, PEG10 – paternally expressed 10, WNV – West Nile Virus. Data points represent the mean ± s.d. (n = 3 independent experiments). P values were calculated using an ordinary unpaired one-sided ANOVA comparing every condition to the ECFP control. *P < 0.05, **P < 0.01. Exact P values: SARS-CoV-2 – 0.001, SARS-CoV-1 – 0.001. F In vivo dual-fluorescence of mutants of SARS-CoV-2 –1PRF RNA in HEK293 cells in the presence and absence of ZAP-S. Datapoints represent the mean ± s.d. (n = 3 independent experiments). P values were calculated using an unpaired one-sided ANOVA comparing values of the ECFP control. * P < 0.05. Exact P values: WT – 0.0003. See also Supplementary Table 2 as well as Fig. 4 for schematics of the mutants used here.