Interferon-induced transmembrane protein 3 (IFITM3) is a critical antiviral protein that restricts infection by a wide array of viruses. This protein localizes to cellular endosomes and lysosomes, where it traps endocytosed viruses within an expanded compartment called the endolysosome. It has been demonstrated that IFITM3 modifies membrane structure and fluidity, and requires an amphipathic helix for its antiviral activity. Importantly, palmitoylation, a lipid posttranslational modification, is also required for IFITM3’s full antiviral activity and could serve as an anchor for this amphipathic helix.
Palmitoylation of cellular proteins is mediated primarily by a family of enzymes called the zinc finger DHHC domain-containing palmitoyltransferases (ZDHHCs), but which members modify IFITM3 are unknown. In this work, we overexpressed each of the 23 mammalian ZDHHCs with IFITM3 and unexpectedly observed that more than half of these enzymes increased IFITM3 palmitoylation with ZDHHCs 3, 7, 15, and 20 having the greatest effect. Amongst these enzymes, ZDHHC20 uniquely increased IFITM3’s antiviral activity when both proteins were overexpressed. In addition, when compared to ZDHHCs 3, 7, and 15, ZDHHC20 uniquely co-localized with IFITM3 in lysosomes which suggest that the location of IFITM3 palmitoylation may influence its activity. However, by screening a library of human cell lines individually knocked-out for each ZDHHC, we found that IFITM3 palmitoylation and restriction of influenza virus was vibrant in the absence of any one ZDHHC, including ZDHHC20. This suggested functional redundancy in the palmitoylation dependent IFITM3-mediated antiviral response. In support of this, siRNA-mediated knockdown of both ZDHHC3 and ZDHHC7 in ZDHHC20 knockout cells decreased endogenous IFITM3 palmitoylation. Overall, our results demonstrate that multiple ZDHHCs can palmitoylate IFITM3 to ensure a robust antiviral response, and that ZDHHC20 may serve as a particularly useful tool for understanding and enhancing IFITM3 activity.
In addition to palmitoylation, another major component of this work includes the identification of IFITM3 as the first confirmed restriction factor of human metapneumovirus (hMPV). hMPV is an important viral respiratory pathogen that causes significant disease in infants, children, and the elderly. Interestingly, hMPV utilizes a bifurcated cellular entry strategy, fusing either with the plasma membrane or, after endocytosis, with the endosomal membrane. Given that IFITM3 only restricts viruses that enter cells through endocytosis we hypothesized that IFITM3 should restrict hMPV, at least in part. Here we found that IFITM3 inhibits hMPV infection to an extent that is similar to cells treated with endocytosis inhibitors. Additionally, by using an IFITM3 variant that accumulates at both the plasma membrane and to endosomes, we observed increased inhibition of hMPV. Fusion assay experiments demonstrated that IFITM3 blocks hMPV F protein-mediated membrane fusion and that restriction of hMPV by IFITM3 was reversed by the membrane destabilizing drug amphotericin B. Conversely, we found that infection by some hMPV strains is enhanced by the endosomal protein Toll-like receptor 7 (TLR7), but that IFITM3 retains the ability to restrict hMPV infection even in cells expressing TLR7. Overall, our results identify IFITM3 as an endosomal restriction factor that limits hMPV infection of cells.