Insights from structural studies of the cardiovirus 2A protein

Figure 1. Representation of cardiovirus cell entry, translation and genome organization Upon cell entry, viral (+) single-stranded RNA is translated in the cytoplasm to produce three large precursor proteins (P1, P2 and P3), which are cleaved by proteases to yield functional proteins. Viral genomic RNA contains VPg and IRES at its 5′ untranslated region for efficient translation. The coding region of the viral RNA also contains Stop-Go and frameshifting sites at the 2A–2B junction, which mediate non-canonical translation events (created with BioRender).

Figure 2. Cardiovirus 2A as an RNA-binding protein and frameshift stimulator (A) X-ray crystal structures of the 2A protein from EMCV (left) and TMEV (right). A zoomed-in view of the conserved ‘arginine loop’ is shown. (B) Cryo-EM structure of EMCV 2A bound to the E. coli small ribosome subunit. The arginine loop is inserted into a helical junction in the 16S rRNA. The interaction involves multiple electrostatic contacts with the ribose phosphate backbone and hydrophobic stacking of the guanidinium groups against each other and an exposed base. (C) The EMCV RNA stimulatory element is predicted to adopt both stem-loop (left) and pseudoknot (right) conformations. Conserved sequence elements important for the interaction are highlighted (yellow, left). Recognition of this element by 2A requires both the main stem (blue, right) and additional base-pairing interactions between conserved elements (red, right).

Figure 3. Cardiovirus 2A as a multifunctional virulence factor (A) Proposed mechanism for inhibition of cap-dependent translation by mimicry of eIF4E-binding protein (4E-BP) and sequestration of eukaryotic initiation factor 4E (eIF4E). However, the C-terminal YxxxxLΦ motif is not structurally conserved between 4E-BP1 and 2A. (B) Proposed mechanism for inhibition of cap-dependent translation by 2A preventing phosphorylation of 4E-BP. (C) Alternative model for translational pathology caused by 2A binding to the small ribosome subunit and competing with translational GTPases. (D) Proposed role for 2A in delivering L protein to the cell nucleus, where it binds to Ran and causes inhibition of nucleocytoplasmic trafficking.