Cis-mediated interactions of the SARS-CoV-2 frameshift RNA alter its conformations and affect function.

The RNA genome of SARS-CoV-2 contains a frameshift stimulatory element (FSE) that allows access to an alternative reading frame through -1 programmed ribosomal frameshifting (PRF). -1PRF in the 1a/1b gene is essential for efficient viral replication and transcription of the viral genome. -1PRF efficiency relies on the presence of conserved RNA elements within the FSE. One of these elements is a three-stemmed pseudoknot, although alternative folds of the frameshift site might have functional roles as well. Here, by complementing ensemble and single-molecule structural analysis of SARS-CoV-2 frameshift RNA variants with functional data, we reveal a conformational interplay of the 5' and 3' immediate regions with the FSE and show that the extended FSE exists in multiple conformations. Furthermore, limiting the base pairing of the FSE with neighboring nucleotides can favor or impair the formation of the alternative folds, including the pseudoknot. Our results demonstrate that co-existing RNA structures can function together to fine-tune SARS-CoV-2 gene expression, which will aid efforts to design specific inhibitors of viral frameshifting.

The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting.

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.