In vitro translation assays in rabbit reticulocyte lysate

In vitro translation reactions were performed using nuclease-treated rabbit reticulocyte lysate (RRL) (Promega) following the manufacturer’s protocol for non-radioactive luciferase reactions, with the following modifications. In vitro translation reactions were pre-incubated with the corresponding recombinant nsp1 variant for 10 min at 4°C before addition of 40nM of the corresponding mRNA. Reactions were then incubated at 30°C for 30 min, whereupon luciferase assays were performed using the NanoLuc luciferase assay kit (Promega). Luminescence was measured using the Spark multimode microplate reader (TECAN). Technical triplicate measurements were taken for each biological replicate. These technical triplicates were averaged to plot for a given biological replicate and normalized to independent luminescence measurements from in vitro translation reactions containing the corresponding concentration of GST in place of nsp1.