Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch

Abstract

Encephalomyocarditis virus 2A protein is a multi functional virulence factor essential for efficient virus replication with roles in stimulating programmed -1 ribosomal frameshifting (PRF), inhibiting cap-dependent translational initiation, interfering with nuclear import and export and preventing apoptosis of infected cells. The mechanistic basis for many of these activities is unclear and a lack of structural data has hampered our understanding. Here we present the X-ray crystal structure of 2A, revealing a novel 'beta-shell' fold. We show that 2A selectively binds to and stabilises a specific conformation of the stimulatory RNA element in the viral genome that directs PRF at the 2A/2B* junction. We dissect the folding energy landscape of this stimulatory RNA element, revealing multiple conformers, and measure changes in unfolding pathways arising from mutation and 2A binding. Furthermore, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a high-resolution cryo-EM structure of 2A bound to initiated 70S ribosomes. In this complex, three copies of 2A bind directly to 16S ribosomal RNA at the factor binding site, where they may compete for binding with initiation and elongation factors. Together, these results provide an integrated view of the structural basis for RNA recognition by 2A, expand our understanding of PRF, and provide unexpected insights into how a multifunctional viral protein may shut down translation during virus infection.

Investigating molecular mechanisms of 2A-stimulated ribosomal pausing and frameshifting in Theilovirus

“…We define a minimal TMEV stimulatory element necessary for 2A binding and show that the protein forms a 1:1 RNA-protein complex with nanomolar affinity. We provide evidence that the alternative pseudoknot-like conformation recently described for the EMCV stimulatory element ( 28 ) is also likely to be present in TMEV and other cardioviruses. Finally, we use metabolic labelling and ribosome profiling to study 2A-stimulated frameshifting and translation of the TMEV genome at sub-codon resolution in infected cells.…”

“…Our previous investigations of protein-stimulated frameshifting in EMCV and TMEV have revealed that the viral 2A protein acts in complex with a stem-loop downstream of the slippery sequence, and that this interaction can be disabled by mutating either a conserved cytosine triplet in the loop or a pair of conserved arginine residues in the protein ( 11 , 22 , 23 ). More recently, we solved the structure of EMCV 2A, revealing a novel protein fold that permits binding to both the stem-loop and to ribosomal RNA with high affinity ( 28 ). However, 2A protein sequences are highly divergent within cardioviruses, and the TMEV protein shares only ∼27% pairwise amino acid sequence identity with its EMCV orthologue.…”

Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

“…As demonstrated by our ensemble and single-molecule analysis, ZAP-S directly interacts with the SARS-CoV-2 frameshift signal and alters folding of the RNA element. However, several trans-acting factors including the cardiovirus 2A and SFL were also shown to bind to ribosomes 15,53 . To explore the possibility of whether ZAP also interacts with ribosomes during translation, we performed ribosome pelleting assay (Fig.…”

“…These derivations were statistically analyzed to identify the folding events and their coordinates. For data fitting, we employed a combination of two worm-like chain models (WLC1 for the fully folded double-stranded parts and WLC2 for the unfolded single-stranded parts) as described previously 53 . Firstly, the initial contour length of the folded RNA was set to 1231 nm, and the persistence length of the double-stranded part was fitted 53 .…”

“…For data fitting, we employed a combination of two worm-like chain models (WLC1 for the fully folded double-stranded parts and WLC2 for the unfolded single-stranded parts) as described previously 53 . Firstly, the initial contour length of the folded RNA was set to 1231 nm, and the persistence length of the double-stranded part was fitted 53 . Then, the persistence length of the unfolded RNA was set to 1 nm, and the contour length of the singlestranded part was fitted.…”