Abstract
Interferon-induced transmembrane protein 3 (IFITM3) is a cellular endosome- and lysosome-localized protein that restricts numerous virus infections. IFITM3 is activated by palmitoylation, a lipid posttranslational modification. Palmitoylation of proteins is primarily mediated by zinc finger DHHC domain-containing palmitoyltransferases (ZDHHCs), but which members of this enzyme family can modify IFITM3 is not known. Here, we screened a library of human cell lines individually lacking ZDHHCs 1-24 and found that IFITM3 palmitoylation and its inhibition of influenza virus infection remained strong in the absence of any single ZDHHC, suggesting functional redundancy of these enzymes in the IFITM3-mediated antiviral response. In an overexpression screen with 23 mammalian ZDHHCs, we unexpectedly observed that more than half of the ZDHHCs were capable of increasing IFITM3 palmitoylation with ZDHHCs 3, 7, 15, and 20 having the greatest effect. Among these four enzymes, ZDHHC20 uniquely increased IFITM3 antiviral activity when both proteins were overexpressed. ZDHHC20 colocalized extensively with IFITM3 at lysosomes unlike ZDHHCs 3, 7, and 15, which showed a defined perinuclear localization pattern, suggesting that the location at which IFITM3 is palmitoylated may influence its activity. Unlike knock-out of individual ZDHHCs, siRNA-mediated knockdown of both ZDHHC3 and ZDHHC7 in ZDHHC20 knock-out 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.
Highlights
Interferon-induced transmembrane protein 3 (IFITM3) is a cellular endosome- and lysosome-localized protein that restricts numerous virus infections
Cells were metabolically labeled with the alk-16 chemical reporter of protein palmitoylation, and immunoprecipitated IFITM3 was subjected to reaction via click chemistry with azidorhodamine to allow fluorescent visualization of palmitoylation by fluorescence gel scanning [5, 32,33,34]
The activity of IFITM3 against influenza virus is negatively regulated by posttranslational modifications, including ubiquitination [6, 18], methylation [46], and phosphorylation [43, 47], palmitoylation is the only known posttranslational modification that increases IFITM3 antiviral activity [5, 18, 20, 31, 48]
Summary
The palmitoyltransferase ZDHHC20 enhances interferoninduced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity. We screened a library of human cell lines individually lacking ZDHHCs 1–24 and found that IFITM3 palmitoylation and its inhibition of influenza virus infection remained strong in the absence of any single ZDHHC, suggesting functional redundancy of these enzymes in the IFITM3-mediated antiviral response. ZDHHC20 enhances IFITM3 palmitoylation ther highlights its critical importance in the function of IFITM3 and other IFITMs. S-Palmitoylation of proteins is typically mediated by members of a family of enzymes known as the zinc finger DHHC (Asp-His-His-Cys) domain– containing palmitoyltransferases (ZDHHCs) (26 –28). We screened overexpressed ZDHHCs and found that numerous members of this enzyme family are able to palmitoylate IFITM3 and that ZDHHC20 is adept at modifying IFITM3 and enhancing its antiviral activity. Unlike individual knock-out cells, knockdowns of additional ZDHHCs in the context of ZDHHC20 KO cells resulted in decreased IFITM3 palmitoylation, confirming a role for ZDHHC20 in palmitoylating endogenous IFITM3
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