Validating DMS Measurements with Dual Reporters

In order to validate DMS measurements, we characterized a series of individual variants using both a fluorescence-based reporter (22 (link)) and a dual-luciferase reporter for −1PRF in the SINV 6K gene. Fluorescence reporters bearing polyprotein variants of interest were transiently expressed in HEK293T cells, and the relative mKate: eGFP intensity ratios were determined by flow cytometry as previously described (22 (link)). HEK293T cells were transiently transfected with dual-luciferase reporter constructs using lipofectamine 3000 (Invitrogen, Carslbad, CA) in accordance with the manufacturer's instructions. Cells were harvested two days post-transfection and lysed using the Passive Lysis Buffer provided in the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). This lysis buffer contains CHAPS detergent that should solubilize any remaining uncleaved, membrane bound reporter enzymes. The relative activities of the firefly luciferase and renilla luciferase domains in the clarified lysate were then measured using the Dual-Luciferase Reporter Assay System on a Synergy Neo2 plate reader (Biotek, Winooski, VT) in accordance with the manufacturer's instructions. To determine the −1PRF efficiency, the firefly: renilla luciferase ratio was normalized relative to that of a construct lacking the insert as was previously described (34 (link)).