Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

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 present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.

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.