Functional dissection of Shiftless identifies features enhancing ribosomal frameshifting inhibition

Programmed ribosomal frameshifting (PRF) is a gene regulatory mechanism essential for the expression of many viral genes. Identifying and optimizing host proteins that interfere with PRF could inform new antiviral strategies. The human protein Shiftless was recently identified as a broad-spectrum, but relatively low-efficacy inhibitor of PRF, yet a comprehensive functional dissection has been lacking, preventing engineering of optimized Shiftless variants. Here, we present an integrated high-throughput screening system that enables quantitative assessment of PRF inhibition by large libraries of protein variants. Using this platform, we performed deep mutagenesis of Shiftless and measured the activity of >5,000 variants in a cell-based dual-fluorescence reporter assay. The resulting mutational landscape revealed clusters of residues critical for PRF inhibition and identified mutations enhancing Shiftless activity against the HIV gag-pol frameshifting site. This approach provides a generalizable framework for dissecting and engineering proteins that regulate alternative translation mechanisms such as PRF and stop-codon readthrough. The authors develop a high-throughput platform to quantify programmed ribosomal frameshifting inhibition by large libraries of protein variants. They identify residues essential for the activity of the human PRF inhibitor Shiftless and mutations that enhance inhibition of HIV gag-pol frameshifting.