Functional redundancy, complementation, and cross-talk between CH25h and IFITM3 in infection and in IFITM3 trafficking and secretion

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 different viruses. When using HIV-1 and vesicular stomatitis virus (VSV), IFITM3 and CH25h restrict viral entry with similar efficiency but act in a largely redundant manner, a finding that is consistent across infection systems, cell types, and entry assays. However, this is not the case for several orthoflaviviruses against which IFITM3, but not CH25h, provides a strong block to infection. We further uncover that the CH25h-25HC axis remodels IFITM3 trafficking by driving partial relocalization of IFITM3 from endolysosomal compartments to the plasma membrane. This occurs by impairing IFITM3 internalization into early endosomes and leads to an increase in its secretion in exosomal vesicles, overall indicating that CH25h contributes to regulate IFITM3 cellular dynamics. Together, these findings reveal that the apparent redundancy of maintaining two distinct effectors targeting the same step of the viral life cycle is not superfluous but instead allows the cell to compensate and, nonetheless, target viruses that may have developed resistance to one of these effectors. Furthermore, the ability of CH25h to remodel IFITM3 trafficking indicates that these two pathways are connected, adding an additional layer of complexity in the biology of IFITM3.IMPORTANCEIFITM3 and CH25h are two potent, broad-acting inhibitors of viral membrane fusion, but their functional connections have not been explored. Here, we show that although their antiviral activities are largely redundant against HIV-1 and vesicular stomatitis virus (VSV), IFITM3 functionally compensates for a relative resistance of orthoflaviviruses toward CH25h. Furthermore, CH25h redistributes IFITM3 from endosomal compartments to the plasma membrane, enhancing its release in exosomal vesicles. Overall, these findings reveal that despite the fact that IFITM3 and CH25h can act redundantly against certain classes of viruses, IFITM3 can functionally compensate for CH25h resistance against other viruses, highlighting why it is important for the cell to maintain two distinct pathways to block virus-to-cell membrane fusion. In addition, these results also reveal how interferon-driven lipid remodeling can reshape the trafficking behavior of IFITM3, leading it to be more present in exosomes, where it could exert paracrine functions that remain to be explored.