A Cascade of Conformational Switches in SARS-CoV-2 Frameshifting: Coregulation by Upstream and Downstream Elements.

Targeting ribosomal frameshifting has emerged as a potential therapeutic intervention strategy against COVID-19. In this process, a -1 shift in the ribosomal reading frame encodes alternative viral proteins. Any interference with this process profoundly affects viral replication and propagation. For SARS-CoV-2, two RNA sites associated with ribosomal frameshifting are positioned on the 5' and 3' of the frameshifting residues. Although much attention has been focused on the 3' frameshift element (FSE), the 5' stem-loop (attenuator hairpin, AH) can play a role. Yet the relationship between the two regions is unknown. In addition, multiple folds of the FSE and FSE-containing RNA regions have been discovered. To gain more insight into these RNA folds in the larger sequence context that includes AH, we apply our graph-theory-based modeling tools to represent RNA secondary structures, "RAG" (RNA-As-Graphs), to generate conformational landscapes that suggest length-dependent conformational distributions. We show that the AH region can coexist as a stem-loop with main and alternative 3-stem pseudoknots of the FSE (dual graphs 3_6 and 3_3 in our notation) but that an alternative stem 1 (AS1) can disrupt the FSE pseudoknots and trigger other folds. A critical length for AS1 of 10-bp regulates key folding transitions. Together with designed mutants and available experimental data, we present a sequential view of length-dependent folds during frameshifting and suggest their mechanistic roles. These structural and mutational insights into both ends of the FSE advance our understanding of the SARS-CoV-2 frameshifting mechanism by suggesting how alternative folds play a role in frameshifting and defining potential therapeutic intervention techniques that target specific folds.