Fig. 5. Analysis of binding of NEIL2 to the 5’-UTR of SARS-CoV-2.

a A549-ACE2 cells were transfected with rNEIL1 or rNEIL2 proteins for 24 h and then infected with CoV-2 (MOI 1.87). Cell extracts were prepared 8 h post infection and subjected to RNA-ChIP analysis using anti-NEIL1 or anti-NEIL2 antibodies or IgG. RT-qPCR was carried out with 5’-UTR, 3’-UTR or gene body specific primers for CoV-2 RNA. Results are represented as % inputs over IgG where error bars show ± standard deviation from the mean; n = 3 independent experiments. p values (unpaired two-tailed Student’s t test) vs. enrichment at gene body. Source data are provided as a Source Data file. b Titration of 1 nM radiolabeled CoV-2 5’-UTR RNA probe with rNEIL2 in an affinity-based binding study as analyzed by RNA-EMSA. c Titration of 100 nM unlabeled CoV-2 5’-UTR RNA traced with radiolabeled RNA probe with rNEIL2 in a stoichiometry-based binding study as analyzed by RNA-EMSA. d Dose-response curve generated by plotting the percentage of bound RNA as shown in b against Log₁₀ of rNEIL2 concentrations for calculation of Hill coefficient using the indicated equation, where bottom = 10, top = 94.42 and EC₅₀ = 46.17 (top and bottom are plateaus in the unit of the y-axis). Titration of 1 nM radiolabeled CoV-2 5’-UTR- ZnF-site-1 (e) and ZnF-site-2 (f) containing RNA probes with rNEIL2 in an affinity-based binding study as analyzed by RNA-EMSA. Dose-response curve were generated by plotting the percentage of bound RNA (bottom panels) against log₁₀ of rNEIL2 concentrations for calculation of Hill coefficient. FP represents free probe. Representative images and quantitation from n = 3 independent experiments with similar results are shown for (b–f).