Fig 1.
Bioactive compounds that affect VZV gB/gH-gL-mediated cell fusion identified using a HT-SRFA.
(A) Schematic of the HT-SRFA with Renilla luciferase dual split protein (DSP). Fusion of effector cells, CHO-DSP1, transiently expressing VZV glycoproteins, gB and gH[TL]-gL, with target cells, MeWo-DSP2, reconstitutes the Renilla luciferase DSP. Effector and target cells were seeded into 384-well plates, and treated with compound libraries for 48 hrs before measuring fusion efficiency; cell viability was assessed by CellTiter-Glo. Positive controls were wells without drug; negative controls had effector cells transfected with empty vectors; medium only controls were included for cell viability. (B) Scatter plot of cell fusion and cell viability derived from the HT-SRFA. The Y-axis and X-axis indicate fusion efficiency and cell viability values normalized to the mean of positive controls. The mean of the percentage (% positive ctrl) from two biological replicates are shown: blue circles are all 4,846 compounds screened, red and black circles are positive and negative controls and the black crosses are medium only. Grey and orange boxes show ±3 CV % of positive controls for fusion efficiency and cell viability respectively. (C) Frequency (%) of compounds that affect cell fusion identified by the HT-SRFA (left), compound classes of fusion enhancers (center) and inhibitors (right), and number of compounds in each class.
Fig 2.
Inhibition of calcineurin phosphatase activity enhances VZV gB/gH-gL mediated cell fusion.
(A) Chemical structures of tacrolimus, pimecrolimus and sirolimus; pimecrolimus group substitutions indicated by red arrows. Regions of interaction with FKBP1A (blue), calcineurin (yellow) and mTOR (red). (B) Schematic of drug interactions with cellular factors. (C and D) Cell fusion and cell viability dose-response curves to tacrolimus, pimecrolimus and sirolimus. CHO-DSP1 or MeWo-DSP1 cells transiently expressing VZV gB/gH[TL]-gL co-cultured with MeWo-DSP2 cells, treated with drug at indicated concentrations. Cell fusion efficiency (C) and cell viability (D) were quantified and normalized to positive controls (medium; no drug). Data are represented as mean ± standard error of the mean (SEM) for ≥3 independent experiments. Dash lines indicate the cutoff for statistically significant enhanced fusion or cytotoxicity. (E) Fluorescence microscopy of GFP-NFATC1 nuclear translocation to demonstrate calcineurin phosphatase activity in MeWo cells. GFP-NFATC1 nuclear translocation induced by ionomycin-triggered calcineurin activation (ionomycin; upper right panel) in MeWo cells and prevention by treatment with tacrolimus (+Tacrolimus) and pimecrolimus (+Pimecrolimus). Nuclei stained with Hoechst 33342 (blue) and GFP-NFATC1 (green). Representative fluorescence microscopy images are shown from three independent experiments. Scale bars = 15 μm. (F) Tacrolimus and pimecrolimus induced binding of FKBP1A and calcineurin in MeWo cells. Western blots of FKBP1A-His (anti-FKBP1A) and calcineurin (anti-calcineurin subunit A; CNA) in eluates from CHO cells transfected with His-tagged FKBP1A or control plasmids that were lysed, precipitated with nickel agarose beads, then mixed with MeWo cell extract and treated with DMSO, tacrolimus (Tac; 10 μM), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM). (G) Box and whisker plots for cell fusion quantified by the SRFA using CHO-DSP1 cells transfected with plasmids expressing either VZV gB/gH[TL]-gL, HSV-1 gB/gH-gL/gD, or syncytin-1 and mixed with MeWo-DSP2, untreated (medium; M) or treated with DMSO (D), pimecrolimus (Pim; 10 μM) or sirolimus (Siro; 10 μM) for 48 hrs. Fusion efficiency was measured and normalized to medium (% medium). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean (+) is from three independent experiments. Statistical differences were evaluated by two-way ANOVA (ns, not significant; *, p < 0.05; ****, p < 0.0001).
Fig 3.
Pimecrolimus disruption of calcineurin-dependent regulation of gB/gH-gL fusion requires FKBP1A.
(A) FKBP1A shRNA knockdown (KD) in MeWo cells. FKBP1A Transcript levels detected by RT-qPCR in MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells were normalized to housekeeping gene PGM1 and presented as a percentage of control cells, with mean ± SEM from three independent experiment (two-tailed, unpaired t-test, ****, p < 0.0001). Western blot of FKBP1A protein (anti-FKBP1A). FKBP1A band density was normalized to α-tubulin in control and FKBP1A KD cells respectively. The ratio of the two values was presented as a percentage of control cells. A representative picture, and mean ± SEM of the ratio from three independent experiments are shown. (B) Quantification of calcineurin phosphatase activity in FKBP1A KD cells using the NFATC1 nuclear translocation assay. MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells transiently expressing GFP-NFATC1 were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min before ionomycin (Iono, 1 μM; 30 min). Fluorescence microscopy images of GFP-NFATC1 (green), nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. Red arrowheads indicate nucleus localization (N), white arrowhead indicates diffused localization in both nucleus and cytoplasm (N/C), and white arrows indicate cytoplasm localization (C). Box and whisker plots of GFP-NFATC1 positive cells. The percentage of GFP positive cells in control and FKBP1A KD cells with GFP-NAFTC1 localization to N, N/C, and C. Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean (+) of cells (n = 15 fields of view, 13–22 cell per field of view) from two independent experiments. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each treatment condition (Untreated, DMSO+Iono, and Pim+Iono) compared to that in the control cells, and significantly different pairs are shown (****, p < 0.0001). (C and D) Effect of FKBP1A KD on VZV gB/gH-gL-mediated fusion and response to pimecrolimus. Box and whisker plots for cell fusion quantified by the SRFA in MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells transfected with plasmids to express VZV gB/gH[TL]-gL and co-cultured with MeWo-DSP2 cells, untreated (medium; Med) or treated with DMSO or pimecrolimus (Pim, 10 μM) for 48 hrs. Cell fusion efficiency when untreated was compared between FKBP1A KD cells and control cells, shown as a percentage of control cells (C), and the response to pimecrolimus was evaluated by normalization to the untreated (% medium) per control and FKBP1A KD cell line respectively (D). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean (+) from three independent experiments. Dots are outliers. Statistical differences were evaluated by unpaired, nonparametric Mann-Whitney test (C) (ns, not significant) or two-way ANOVA (D) (ns, not significant; ****, p < 0.0001).
Fig 4.
Calcineurin supports VZV gB/gH-gL mediated cell fusion.
(A) CNB1 shRNA knockdown (KD) in MeWo cells. CNB1 transcript levels in MeWo-DSP1 control cells or CNB1 KD MeWo-DSP1 cells detected by RT-qPCR were normalized to housekeeping gene PGM1 and presented as a percentage of control cells, with mean ± SEM from two independent experiment (two-tailed, unpaired t-test, ****, P < 0.0001). Western blot of CNB1 protein (anti-CNB1). CNB1 band density was normalized to α-tubulin in control and CNB1 KD cells respectively. The ratio of the two values was presented as a percentage of control cells. A representative picture, and mean ± SEM of the ratio from two independent experiments are shown. (B and C) Effect of CNB1 KD on VZV gB/gH-gL-mediated fusion and response to pimecrolimus. Box and whisker plots for cell fusion quantified by the SRFA in MeWo-DSP1 control cells or CNB1 KD MeWo-DSP1 cells transfected with plasmids to express VZV gB/gH[TL]-gL and co-cultured with MeWo-DSP2 cells, untreated (medium; Med) or treated with DMSO or pimecrolimus (Pim, 10 μM) for 48 hrs. Cell fusion efficiency when untreated was compared between CNB1 KD cells and control cells, shown as a percentage of control cells (B), and the response to pimecrolimus was evaluated by normalization to the untreated (% medium) per control and CNB1 KD cell line respectively (C). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean (+) from two independent experiments. Statistical differences were evaluated by unpaired, nonparametric Mann-Whitney test (B) (**, p < 0.01) or two-way ANOVA (C) (ns, not significant; *, p < 0.05; ****, p < 0.0001).
Fig 5.
Calcineurin phosphatase activity remains functional in VZV infected cells.
(A) Fluorescence microscopy images of MeWo cells transiently expressing GFP-NFATC1 at 16 hpi with pOka-TK-RFP that were untreated (medium) or treated with DMSO or pimecrolimus (Pim, 10 μM) 30 min prior to ionomycin stimulation (Iono, 1 μM; 30 min); pOka-TK-RFP (red), GFP-NFATC1 (green), and nuclei stained with Hoechst 33342 (blue), and a composite image. Scale bars = 15 μm. (B) Box and whisker plots of GFP-NFATC1 localization in VZV infected cells. Of the total GFP-NFATC1 expressing cells that were also infected with TK-RFP, the percentage of cells with GFP-NAFTC1 translocated to nucleus (N), diffused in nucleus and cytosol (N/C), or localized in cytosol (C). Boxes represent 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean (+) of cells (n = 12 fields of view, 41–66 cell per field of view) from three independent experiment. Dots are outliers. Statistical difference were analyzed by two-way ANOVA for each localization group (N, N/C, and C) compared to that in the untreated, shown are the significantly different pairs (****, p < 0.0001).
Fig 6.
Enhanced cell fusion induced by inhibition of calcineurin phosphatase activity during VZV infection.
(A) Confocal microscopy of LifeAct-tGFP MeWo cells infected with pOka-TK-RFP, treated with DMSO or pimecrolimus (Pim; 10 μM). Representative plaques captured from live-cell confocal microscopy at 24 and 30 hpi from S1 and S2 Movies, that show pOka-TK-RFP infected cells (red), LifeAct-tGFP labeled actin filaments (green), and nuclei stained with Hoechst 33342 (blue). Scale bars = 100 μm. (B) Frequency of nuclei in syncytia at 40 min intervals from 24 to 30 hpi for plaques in (A). Coefficient of determination (R2) was calculated to determine the linear relationship of increased nuclei frequency in syncytia during the 6 hour window of infection. (C) Scatter dot plots of increased nuclei frequency in syncytia from 24 to 30 hpi. Mean ± SEM for plaques (n = 8) from two independent live-cell confocal microscopy experiment. Statistical significance was evaluated by an unpaired, nonparametric Mann-Whitney test (*, p < 0.05).
Fig 7.
Inhibition of calcineurin phosphatase activity suppresses VZV spread in MeWo cells.
Plaque sizes of VZV pOka in MeWo cells untreated (medium; Med), treated with DMSO or pimecrolimus (Pim; 10 μM) at 4 dpi. (A) Immunohistochemistry staining of VZV plaques with the mean plaque size per condition. Scale bars = 0.3 mm. (B) Box and whisker plots of plaque sizes (mm2). Boxes show the 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean (+) from three independent experiments (n = 60). Dots are outliers. Statistical differences were evaluated by one-way ANOVA (ns, not significant; ****, p < 0.0001). (C) Box and whisker plots of MeWo cells, MeWo-DSP1 control cells or FKBP1A KD MeWo-DSP1 cells at 4 dpi with VZV pOka, untreated (medium; Med) or treated with DMSO or pimecrolimus (Pim, 10 μM). Percentage of plaque size normalized to the untreated (% medium). Boxes show the 25–75 percentile, whiskers extend to 10–90 percentile, the median is the horizontal band, and the mean (+) from two independent experiments (n = 60). Dots are outliers. Statistical differences were evaluated by two-way ANOVA (ns, not significant; ****, p < 0.0001). (D) Frequency and total area of plaques in the presence of pimecrolimus. MeWo cells infected with pOka were untreated (medium), treated with DMSO or pimecrolimus (10 μM) for 4 days, plaque frequency and total plaque area were analyzed by ImageJ automatic particle counting. Representative result from ≥3 independent experiments is shown, with input Immunohistochemistry images (scale bars = 2 mm), processed images and measurements listed.
Fig 8.
Inhibition of calcineurin phosphatase activity alters the phosphoproteome of MeWo cells.
(A) Consensus docking motifs on calcineurin substrates for calcineurin binding. PxIxIT and LxVP motifs from 20 previously verified substrates of calcineurin were used to generate the consensus motif by WebLogo 3. Frequency of amino acids at each position is represented by the height of the letter. Chemical properties of amino acids are represented by the color of the letter: green, polar; purple, neutral; blue, basic; red, acidic; black, hydrophobic. (B) Heat map of uniquely phosphorylated proteins detected in MeWo cells treated with pimecrolimus (10 μM) for 2 hrs. Total cellular proteins were extracted in the presence of phosphatase inhibitor and subject to Zr-IMAC phosphopeptide enrichment, followed by orbitrap mass spectrometry. Three biological samples were analyzed for each condition and the relative spectra of uniquely phosphorylated proteins detected in all three pimecrolimus treated samples but not in DMSO samples are shown in the heat map. Nuclear factor of activated T cells cytoplasmic 1 (NFATC1); ETS transcription factor (ELK1); phosphatase and actin regulator 2 (PHACTR2); interleukin enhancer binding factor 3 (ILF3); nuclear receptor coactivator 1 (NCOA1); transmembrane channel-like protein 8 (TMC8); methyl-CpG binding domain protein 1 (MBD1). Previously known calcineurin substrates are highlighted with asterisks (*). (C) Potential docking motif for calcineurin binding on newly identified phosphoproteins located by Clustal alignment using the consensus motif. Conserved amino acids on PxIxIT and LxVP motif (red), conservative substitutions (blue), docking motif on previously known substrates of calcineurin (boxed).