Structural insights into West Nile virus programmed ribosomal frameshifting reveal coordinated regulation by RNA, ribosome, and nascent peptide

Programmed -1 ribosomal frameshifting (-1 PRF) is essential for West Nile virus (WNV) gene expression and generates NS1', a protein implicated in viral replication and pathogenesis. However, the structural pathway by which translating ribosomes traverse the WNV frameshift signal remains poorly understood. Here, we combined cryo-electron microscopy, RNA structural modeling, molecular dynamics simulations, and functional analyses to investigate the mechanism of WNV -1 PRF. We resolved a pre-frameshift ribosomal complex together with four translocation intermediates, including a previously uncharacterized semi-rotated state in which the 40S subunit undergoes partial rotation while tRNAs remain in near-canonical A/A-P/P configurations. This intermediate is accompanied by remodeling of conserved peptidyl-transferase-center nucleotides, suggesting that active-site rearrangements can precede canonical tRNA movement during translocation. Structural modeling and simulations further suggest that the WNV frameshift-stimulatory RNA exists as a compact but conformationally heterogeneous ensemble capable of adopting multiple stable or metastable pseudoknot architectures. In addition, we identified direct interactions between the nascent viral peptide and the ribosomal exit tunnel, and disruption of the tunnel-occupying peptide reduced frameshifting efficiency. Together, our findings suggest that WNV frameshifting is governed not by a single stimulatory element but by the coordinated coupling of RNA dynamics, ribosome remodeling, and nascent-chain interactions.IMPORTANCEViruses often rely on a process called programmed ribosomal frameshifting to produce proteins important for infection. West Nile virus (WNV), a mosquito-borne pathogen that can cause severe neurological disease in humans, uses this strategy to generate NS1', a protein linked to viral replication and pathogenesis. However, how ribosomes are redirected into an alternative reading frame remains incompletely understood. Here, we provide structural insights into ribosome movement during translation of the WNV frameshift signal. Our findings also suggest that the viral RNA can adopt alternative conformations and that the emerging viral peptide may influence frameshifting efficiency. Together, these observations contribute to our understanding of how RNA structure, ribosomal dynamics, and nascent peptides may influence viral protein expression.