The virus uses an 18-residue peptide sequence (so-called P2A peptide) that trips up the ribosome, causing translation to skip over this sequence and express the next ORF, thereby producing multiple separate proteins at near equimolar amounts. The authors validated this system using a construct wherein firefly luciferase (FLuc) and Renilla luciferase (RLuc) were separated by the P2A peptide sequence under control of a CRE response element (Figure). Coincidence expression of both reporters using this construct in HEK293 showed near equal expression of FLuc and RLuc as separate proteins with little detectable fusion protein (Figure).
In the assay, activity from both reporters can be measured in the same assay well using standard detection reagents (e.g., DualGlo, Promega). A small library of known drugs was screened (the Library of Pharmacology Active Substance; LOPAC, Sigma) and it was demonstrated that compounds annotated as agonists of CRE-dependent signaling were identified as active with both FLuc and RLuc signals; however, compounds known to specifically inhibit either FLuc or RLuc were only found when measuring the activity of the reporter that was inhibited. Many reporter inhibitors actually lead to increased reporter signal in RGAs because reporter inhibitors can act in cells to stabilize the reporter enzyme, leading to increased enzyme levels that mimic gene activation. This counter-intuitive result can greatly complicate the interpretation of compound activity arising from RGAs, but the coincidence expression demonstrated here rapidly flagged these compounds, which were detected as active in only one of the reporter responses (see also www.reportergene.com).
This system should greatly facilitate the production of orthogonal RGAs as well as other assay systems in which production of multiple proteins is required.