Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch
Programmed –1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein: a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here, we show that 2A binds directly to the frameshift-stimulatory element in the viral RNA with nanomolar affinity and equimolar stoichiometry, and we define the minimal RNA element required for binding. Through site-directed mutagenesis and the use of single-molecule optical tweezers, we study the dynamics of EMCV RNA (wild type- CCC as well CUC mutant), both alone and in the presence of 2A. By observing short-lived intermediate states in real-time, we demonstrate that the EMCV stimulatory element exists in at least two conformations and 2A binding stabilises one of these, an RNA pseudoknot, increasing the force required to unwind it. Finally, we report a direct interaction of 2A with both mammalian and bacterial ribosomes.