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The interferon (IFN)-induced transmembrane protein 3 (IFITM3) is a cellular restriction factor that inhibits infection by influenza virus and many other pathogenic viruses. IFITM3 prevents endocytosed virus particles from accessing the host cytoplasm although little is known regarding its regulatory mechanisms. Here we demonstrate that IFITM3 localization to and antiviral remodeling of endolysosomes is differentially regulated by S-palmitoylation and lysine ubiquitination. Although S-palmitoylation enhances IFITM3 membrane affinity and antiviral activity, ubiquitination decreases localization with endolysosomes and decreases antiviral activity. Interestingly, autophagy reportedly induced by IFITM3 expression is also negatively regulated by ubiquitination. However, the canonical ATG5-dependent autophagy pathway is not required for IFITM3 activity, indicating that virus trafficking from endolysosomes to autophagosomes is not a prerequisite for influenza virus restriction. Our characterization of IFITM3 ubiquitination sites also challenges the dual-pass membrane topology predicted for this protein family. We thus evaluated topology by N-linked glycosylation site insertion and protein lipidation mapping in conjunction with cellular fractionation and fluorescence imaging. Based on these studies, we propose that IFITM3 is predominantly an intramembrane protein where both the N and C termini face the cytoplasm. In sum, by characterizing S-palmitoylation and ubiquitination of IFITM3, we have gained a better understanding of the trafficking, activity, and intramembrane topology of this important IFN-induced effector protein.

Significance: Cysteine fatty acylation (S-fatty acylation) regulates the stability, trafficking, and activity of proteins in eukaryotes. Current methods to study S-fatty acylation rely on the metabolic incorporation of fatty acid analogs or selective chemical labeling and affinity purification, which limits the detection of endogenous S-fatty acylated protein isoforms and levels. Here we demonstrate that the biochemical exchange of acyl groups on cysteines with defined mass-tags enables the direct visualization of endogenous S-fatty acylated protein levels. The application of this mass-tag labeling method revealed that site-specific S-fatty acylation of interferon-induced transmembrane protein 3 controls the antiviral activity of this IFN-induced effector.