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

Fig. 3. Effect of zinc-finger antiviral protein (ZAP) on 1a/1b –1 frameshifting in vitro.

Fig. 3

A The strategy of the in vitro translation assay using rabbit reticulocyte lysate (RRL) and the experimental workflow to study ribosome association of ZAP-S. B Schematics of the N-terminal FLAG-tagged frameshifting reporter consisting of the nucleotides 12686-14190 (~1.5 kb) of the SARS-CoV-2 genome. RNAs were translated in RRL in the presence of increasing concentrations of ZAP-S ranging from 0 to 3 µM. FLAG-tagged peptides generated by ribosomes that do not frameshift (no –1PRF) or that enter the −1 reading frame (−1PRF) were identified via western blotting using anti-DDDDK antibody. FE was calculated as previously described11, by the formula: Intensity (–1-frame)/ (Intensity (–1-frame) + Intensity (0-frame)). Size markers - M (Marker), –1PRF M (–1-frame marker), and no –1PRF M (0-frame marker). n = 3 independent experiments. C Changes in FE observed in the presence of ZAP-S from (B) (normalized to 0 µM ZAP as shown in B). P values were calculated using an ordinary unpaired one-sided ANOVA comparing every concentration to the no ZAP control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Exact P values: 0.25 µM – 0.82, 0.50 µM – 0.26, 0.75 µM – 0.06, 1.00 µM – 0.009, 1.50 µM – 0.0002, 2.00 µM –<0.0001, 3.00 µM–< 0.0001. See also Supplementary Fig. 2and Supplementary Table 3. D Polysome profiling analysis of ZAP-S in RRL. RRL translating the FLAG-tagged SARS-CoV-2 frameshifting reporter was subjected to 5–45% sucrose gradient ultracentrifugation, and subsequently fractionated. Levels of RPL4, as well as ZAP in each fraction, were analyzed by western blotting using anti-RPL4 and anti-ZC3HAV1 (ZAP) antibodies. n = 2 independent experiments. E Ribosome pelleting of untreated Calu-3 cells. Naïve Calu-3 cells were lysed and loaded onto sucrose cushions. Levels of RPL4, ZAP, and β-actin in the pellets were analyzed by western blotting using anti-RPL4, anti-ZC3HAV1 (ZAP) and anti-β-actin antibodies. n = 3 independent experiments. F Polysome profiling analysis of ZAP-S in cells. HEK293 cells transiently expressing ZAP-S were lysed, subjected to 5–45% sucrose gradient ultracentrifugation, and subsequently fractionated. Levels of ribosomal proteins, ZAP as well as SHFL in each fraction, were analyzed by western blotting using anti-RPL4, anti-ZC3HAV1 (ZAP) and anti-RYDEN (SHFL) antibodies. n = 3 independent experiments.