Cyclosporines Antagonize the Antiviral Activity of IFITMProteins by Redistributing Them toward the Golgi Apparatus

Figures

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  Figure 2. Cyclosporine A rescues virus fusion by relocalizing IFITM3 and not by inducing its degradation. A549.Vector (A) and A549.IFITM3 (B) cells were incubated with DMSO or 20 µM CsA or CsH for 1.5 h before adding the BlaM-Vpr containing pseudoparticles for virus fusion measurements. Data are means and S.D. of two independent combined experiments, each performed in triplicate. Statistical analysis was performed using one-way ANOVA. For Western blot analysis, A549.IFITM3 were incubated with DMSO or 20 µM CsA or CsH for 1.5 h (C) or 16 h (D), and total cell lysate was analyzed. Note that Western blots shown in (C,D) are representative experiments; the average numbers and S.D. for normalized IFITM band intensities from 3 independent experiments are provided in the tables beneath the blots. The effect of 1.5 h pretreatment with CsA appears significant due to the inclusion of one outlier experiment. A549.IFITM3 cells were incubated with DMSO or 20 µM CsA or CsH for 1.5 h (E–G), fixed, permeabilized, and stained for IFITM3. For each sample, 3 image fields were acquired. The area covered by IFITM3′s signal per cell was determined from the images using ImageJ, after thresholding, creating an…

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  Figure 1. Cyclosporines A and H inhibit IFITM3 antiviral activity. (A) Scatter plot of screening results of 1280 compounds LOPAC library at 20 µM final concentration. The screening was performed using BlaM assay for IAVpp fusion in MDCK.IFITM3 cells seeded in 384-well plates. The results were normalized to IAVpp fusion in MDCK.Vector cells used as a control for efficient fusion of IAVpp (see also Table S1). Dose–response on IAVpp fusion and cell viability for CsA (B,C) and CsH (D,E) in A549.Vector and A549.IFITM3 cells. Target cells were treated with cyclosporines or DMSO for 1.5 h at 37 °C prior to IAVpp binding. Data are means and S.D. of two independent combined experiments, each performed in triplicate. Statistical analysis was caried out using Student’s t-test. * p < 0.05; ** p < 0.01; ns, not significant.

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  Figure 3. Cyclosporine A relocates IFITM3 toward the Golgi apparatus. A549-IFITM3 cells were incubated with DMSO or CsA (20 µM) for 1.5 h, fixed, permeabilized, and stained for respective proteins. For each sample, 5 image fields were acquired. The JaCoP ImageJ plugin was used to calculate IFITM3, TGN46, and GM130 colocalization using both Pearson’s and Mander’s coefficient. Statistical analysis was performed using Student’s t-test. **, p < 0.01; *** p < 0.001. See also Figures S1 and S2.

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  Figure 4. Cyclosporine A induces IFITM3 relocalization irrespective of the N-terminal endosome-sorting signal. A549 cells transduced with wild-type IFITM3, IFITM3 Y20E, IFITM3 NΔ21, or IFITM1 were incubated with CsA (20 µM) for 1.5 h, fixed, and stained for respective protein together with Golgi marker GM130. For each sample, 3 image fields were acquired. The JaCoP ImageJ plugin was used to measure respective IFITM and GM130 colocalization. Since expression of IFITM1 and IFITM3 mutants was not homogenous across the acquired images, we decided to compare IFITM and GM130 colocalization using Mander’s correlation (% of IFITM signal overlapping with GM130 signal) instead of Pearson’s correlation (relationship between signals) for this and other images. Statistical analysis was performed using Student’s t-test. *** p < 0.001.

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  Figure 5. Cyclosporine-A-mediated IFITM3 relocalization toward the Golgi is completed within 45 min. A549 cells transduced with either IFITM3 or IFITM1 were incubated with CsA (20 µM) for indicated time, fixed, and stained for respective protein together with Golgi marker GM130. (A–E) Images of IFITM3 and Golgi signals after the indicated time of pretreatment with CsA or DMSO (control). (F) Analysis of colocalization of IFITM3 and Golgi markers from panels A–E. (G–K) Images of IFITM1 and Golgi signals after the indicated time of pretreatment with CsA or DMSO (control). (L) Analysis of colocalization of IFITM3 and Golgi markers from panels G–K. For each sample, 5 image fields were acquired. The JaCoP ImageJ plugin was used to measure respective IFITM and GM130 colocalization.

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  Figure 6. Cyclosporine A induces IFITM3 degradation in HeLa cells. HeLa.Vector (A) and HeLa.IFITM3 (B) cells were incubated with DMSO or 20 µM CsA, or CsH for 1.5 h before adding BlaM-Vpr containing pseudoparticles for virus–cell fusion measurements. Data are means and S.D. of two independent combined experiments, each performed in triplicate. Statistical significance was assessed using one-way ANOVA. For Western blot analysis, HeLa cells were incubated with DMSO or 20 µM CsA or CsH for 1.5 h (C), 4 h (D), or 16 h (E), and total cell lysate was analyzed. For immunostaining analysis, HeLa cells were incubated with DMSO or 20 µM CsA or CsH for 1.5 h, fixed, permeabilized, and stained for IFITM3 (F). For each sample, 3 image fields were acquired. The JaCoP ImageJ plugin was used to measure respective IFITM and GM130 colocalization. HeLa cells were treated with 500 units of interferon-alpha for 24 h followed by DMSO, CsA, or CsH treatment for 1.5 h (G) or 4 h (H) and whole-cell lysate was analyzed by Western blotting. Densitometry analysis was performed using ImageJ for three independent experiments. Statistical significance was determined using Student’s t-test. * p < 0.05. For IFN-treated…

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  Figure 7. Cyclosporine-A-driven rescue of viral fusion is through IFITM3 antagonism. HeLa-TKO.Vector (A) and HeLa-TKO.IFITM3 (B) cells were incubated with with DMSO or 20 µM CsA or CsH for 1.5 h before adding BlaM-Vpr containing pseudoparticles for virus–cell fusion measurements. Data are means and S.D. of two independent combined experiments, each performed in triplicate. Statistical significance was assessed using one-way ANOVA. For Western blots, HeLa-TKO.IFITM3 was incubated with DMSO or 20 µM CsA or CsH for 1.5 h (C) or 4 h (D), and total cell lysate was analyzed. The mean-normalized band intensities and S.D. from three independent experiments are shown under the gels. (E) HeLa-TKO and HeLa-TKO.IFITM3 cells were treated as indicated previously, fixed, and stained for IFITM3 and GM130. For each sample, 3 image fields were acquired. The JaCoP ImageJ plugin was used to measure respective IFITM and GM130 colocalization. Statistical analysis was performed using Student’s t-test. ** p < 0.01; *** p < 0.001; ns, not significant.