The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity

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.

Keywords: DHHC; IFITM; IFITM3; ZDHHC; influenza virus; innate immunity; interferon; palmitoyltransferase; post-translational modification (PTM); protein palmitoylation.

Figure 1.

Knock-out of individual human ZDHHCs does not affect IFITM3 palmitoylation or cellular susceptibility to influenza virus infection. A, IFITM3 expression in HAP1 WT and IFITM3 KO cells with or without overnight treatment with IFNβ. B, HAP1 WT and IFITM3 KO treated as in A were infected with influenza A virus (m.o.i., 1) for 24 h. Cells were stained with anti-influenza virus nucleoprotein antibodies to measure percentage of infection by flow cytometry. Average infection percentages of triplicate samples from a representative experiment of more than five experiments were graphed. Error bars represent S.D. C, the indicated HAP1 cell lines were treated with IFNβ overnight to induce expression of IFITM3. Cells were labeled for 1 h with 50 μm alk-16 and lysed prior to immunoprecipitation of IFITM3 and reaction with azidorhodamine (az-rho) via click chemistry for visualization of palmitoylation by fluorescence gel scanning. Western blotting for IFITM3 served as a loading control. The bar graph indicates normalized palmitoylation fluorescence averaged from five independent experiments. Error bars indicate S.D. of the mean. D, the indicated HAP1 cell lines were treated with IFNβ overnight or left untreated. The cells were subsequently infected with influenza A virus (m.o.i., 1) for 24 h. Cells were stained with anti-influenza virus nucleoprotein antibodies to measure percentage of infection by flow cytometry. Average infection percentages from four independent experiments, each performed in triplicate, were graphed. Error bars represent S.D.

Figure 2.

Multiple ZDHHCs can palmitoylate IFITM3. A, HEK293T cells were cotransfected with murine HA-IFITM3 and the 23 HA-tagged murine DHHC constructs as indicated or control vector expressing GST (−). Parentheses indicate the modern ZDHHC nomenclature for these constructs. Cells were labeled for 1 h with 50 μm alk-16. IFITM3 was immunoprecipitated and subjected to reaction with azidorhodamine (az-rho) via click chemistry for visualization of palmitoylation by fluorescence gel scanning. Western blotting with anti-HA antibodies provided a loading control for IFITM3 and showed expression of the ZDHHCs. B, average quantitation of palmitoylation fluorescence normalized for protein loading from four independent experiments was graphed. IFITM3 palmitoylation when coexpressed with the GST control was normalized to a value of 1. Error bars indicate S.D. Asterisks indicate that palmitoylation was increased on average by greater than 1.7-fold over the control. Conditions indicated with an asterisk are also significantly different from the GST control as determined by Student's t test with p < 0.05 in all cases.

Figure 3.

ZDHHC20 overexpression increases IFITM3 antiviral activity. A, HEK293T cells were cotransfected with the indicated HA-tagged ZDHHCs or GST control plus either vector control or myc-IFITM3. ZDHHA20 refers to the ZDHHC20 C156A catalytic mutant. After overnight transfection, cells were infected with influenza virus (m.o.i., 2.5) for 6 h, collected, and stained with anti-influenza nucleoprotein, anti-myc, and anti-HA to determine percentage of infection in the transfected cells by flow cytometry. Data shown are average infection percentages from at least three independent experiments, each done in triplicate. Error bars represent S.D. of the mean. The asterisk indicates p < 0.01, Student's t test. B, HEK293T cells were transfected with the indicated plasmids and labeled for 1 h with 50 μm alk-16. Anti-HA immunoprecipitation of cell lysates followed by reaction with azidorhodamine (az-rho) via click chemistry allowed visualization of palmitoylation by fluorescence gel scanning. Western blots served as a loading control for IFITM3 and as a control for expression of both WT and mutant HA-ZDHHC20.

Figure 4.

ZDHHCs 20 and 7 can palmitoylate all three of IFITM3's cysteines but localize distinctly. A, HEK293Ts were cotransfected with either GST-expressing control vector or HA-ZDHHC20 and HA-IFITM3 or cysteine mutants lacking specific palmitoylation sites. Cells were then labeled with 50 μm alk-16, and immunoprecipitated IFITM3 was subjected to reaction with azidorhodamine (az-rho) via click chemistry for visualization of palmitoylation by fluorescence gel scanning. Anti-HA Western blotting confirmed comparable loading and the expression of the ZDHHCs. B, same as in A except using HA-ZDHHC7. C and D, same as in A and B except using IFITM3 constructs with the indicated truncations. E, palmitoylation signals from C and D were quantified and normalized relative to the anti-HA loading control for IFITM3 expression. For each IFITM3 construct, the normalized palmitoylation signal in the presence of the overexpressed ZDHHC was divided by the palmitoylation signal for the respective GST control. An average of results from four individual experiments is graphed. Error bars represent S.D. The asterisk indicates p < 0.0001, Student's t test. F, confocal microscopy of MEFs transfected overnight with the indicated HA-tagged ZDHHCs. Green represents HA staining (ZDHHCs), and blue represents DAPI (nuclei).

Figure 5.

ZDHHC20 colocalizes extensively with IFITM3. A, MEFs were transfected overnight with the indicated HA-ZDHHC constructs and stimulated with IFNα for 8 h to induce IFITM3 expression. Cells were then stained with anti-HA (ZDHHC), anti-IFITM3, and DAPI for confocal microscopy imaging. B, overlap of IFITM3 with each ZDHHC was quantified from images as in A using the JACoP plugin for ImageJ software. At least 30 cells from two independent experiments were quantified and averaged. Error bars represent S.D. of the mean. Statistical significance for differences between ZDHHC20 and each of the other three ZDHHCs was determined by Student's t test, p < 0.0001. C, MEFs were transfected overnight with HA-ZDHHC20 construct and then stimulated with IFNα for 8 h to induce IFITM3 expression. Cells were then stained with anti-HA (ZDHHC), anti-IFITM3, anti-LAMP1, and DAPI for confocal microscopy imaging. Merge (3) and Merge (4) indicate three- and four-color merged images, respectively.

Figure 6.

Depletion of ZDHHCs 3 and 7 in ZDHHC20 knock-out cells decreases IFITM3 palmitoylation. A–C, ZDHHC20 KO HAP1 cells were transfected with siRNAs against the indicated ZDHHCs or with negative control siRNAs. Cells were also treated with IFNβ overnight to induce expression of IFITM3. A, cells were labeled for 1 h with 50 μm alk-16 and lysed prior to immunoprecipitation of IFITM3 and reaction with azidorhodamine (az-rho) via click chemistry for visualization of palmitoylation by fluorescence gel scanning. Western blotting for IFITM3 served as a loading control. B, graphs represent knockdown efficiency of the indicated siRNAs from a representative experiment as determined using quantitative RT-PCR for the specific ZDHHC transcripts normalized to GAPDH transcript levels. The asterisk indicates p < 0.0001, Student's t test, compared with the control siRNA-treated samples. C, the bar graph indicates normalized palmitoylation fluorescence averaged from three independent experiments. Error bars indicate S.D. The asterisk indicates p < 0.0001, Student's t test. Cont, control.