Functional mapping of IFITM3 resolves its antiviral core and deciphers human influenza susceptibility

Genetic variation in interferon-induced transmembrane protein 3 (IFITM3) contributes to inter-individual differences in influenza outcome, yet a comprehensive functional map of IFITM3 variation remains unavailable. Here we show a high-resolution functional atlas of 2,898 IFITM3 variants across three influenza A strains using deep mutational scanning. Our data reveals a functionally constrained central region, providing functional evidence consistent with a Type II membrane topology and highlighting structural features associated with antiviral activity. Comparison with AlphaMissense reveals systematic limitations in predicting the effects of variants within unstructured N-terminal motifs and lipid-embedded domains. Crucially, computational models fail to recognize variants with enhanced antiviral activity, such as G133Q. By integrating this atlas with ClinVar and gnomAD, we provide a functional readout for rare human alleles that may influence susceptibility to infection. These findings provide a resource for interpreting human variation and establish a framework for studying host-directed antiviral mechanisms. This study generates a high-resolution functional atlas of 2,898 IFITM3 variants using deep mutational scanning. The resulting map resolves the protein’s intramembrane antiviral core and identifies key rare human alleles that dictate susceptibility to influenza infection