Cholesterol Remodeling by CH25h Rewires IFITM3 Trafficking and Secretion Without Enhancing Antiviral Restriction

Type I interferon induces multiple protein effectors to inhibit viral replication. Among these, the interferon-induced transmembrane protein (IFITM3) and the cholesterol-25-hydroxylase (CH25h) converge on the inhibition of viral entry by altering the behavior of membranes. Here, we dissect the functional and mechanistic relationship between these two membrane-acting effectors using HIV-1 and vesicular stomatitis virus (VSV) as models. We show that IFITM3 and CH25h restrict viral entry with similar efficiency, but act in a largely redundant manner during infection. Their redundancy is consistent across infection systems, cell types, and entry assays, indicating that both factors converge on a shared biophysical block to membrane fusion. Unexpectedly, we uncover a second layer of interplay in which the CH25h-25HC axis remodels IFITM3 trafficking. Exposure to 25-hydroxycholesterol drives IFITM3 from endolysosomal compartments to the plasma membrane, impairs its internalization into early endosomes, and increases its secretion in exosomal vesicles. These effects occur independently of direct antiviral activity and reveal that CH25h regulates IFITM3 cellular dynamics. Together, our study identifies a previously unrecognized cross-regulatory circuit between two IFN-induced antiviral pathways, highlighting how lipid remodeling by CH25h, and potentially by other cellular factors, can contribute to control the behavior of IFITM3. ImportanceType I interferons induce numerous antiviral factors that frequently target the same vulnerable steps of viral infection, but how these factors influence one another is not well understood. IFITM3 and the CH25h are two potent, broad-acting inhibitors of viral membrane fusion. Here, we show that although their antiviral activities are largely redundant against HIV-1 and VSV, CH25h profoundly alters the biology of IFITM3. The product of the enzymatic activity of CH25h, the oxysterol 25HC, redistributes IFITM3 from endosomal compartments to the plasma membrane and enhances its release in exosomal vesicles-- effects independent of direct antiviral activity. These findings reveal an unexpected layer of cross-regulation between lipid-modifying enzymes and membrane-embedded restriction factors. More broadly, they highlight how interferon-driven lipid remodeling can reshape the trafficking and secretion of antiviral proteins, expanding the functional landscape of innate immunity beyond viral entry inhibition.