S C-terminal mutations that decrease ezrin interaction enhance transduction by pseudotyped particles in Vero E6 cells.

<p>A. Generation of lentiviral pseudotyped particles harboring wild-type (wt) or mutated (Δ8 and Δ8 C1) SARS-CoV Spike proteins. A Western blot assay was performed on concentrated SARSpp particles where the Spike protein and the lentiviral backbone protein p24 were probed. Estimation of protein quantities was performed using densitometry analysis. B. Entry of wt and mutated SARS-CoV S pseudotyped particles. Vero E6 cells were infected by wt S SARS-CoV pseudotyped particles, along with mutated SARSpp (SΔ8 SARSpp and SΔ8 C1 SARSpp). Results are expressed as fold-change in luciferase activity compared to the wt S SARSpp. The results are averages of triplicates and are representative of at least three independent experiments. * indicates a value of <i>p</i><0.05 in a two-tailed t-test.</p

Characterization of interactions determinants of S endodomain binding to ezrin.

<p>A. Sequences of the wild type (wt) and mutated S endodomain used in the GST-pull down analysis. Twelve GST fusion proteins were produced with mutations in the S endodomain. Δ indicates truncations and C indicates cysteine to alanine mutations (in bold) of cysteine clusters (1 to 4). B. & C. Effect of truncations or cysteine to alanine mutations. Vero E6 lysate was incubated with Glutathione-Sepharose beads coupled or not with GST, GST-S_endo_wt (B. & C.), GST-S_{endo Δ8}, and GST-S_{endo Δ19}(B.), GST-S_{endo C1}, GST-S_{endo C2}, GST-S_{endo C3}, GST-S_{endo C4}, and GST-S_{endo C1-4} (C.) using 1 or 5 µg of GST fusion proteins. D. Effects of cysteine to alanine mutations and truncations or point mutations (K1227A and T1220A). Vero E6 lysate was incubated with Glutathione-Sepharose beads coupled with either GST, GST-S_{endo wt}, GST-S_endo_{Δ8 C1-4}, and GST-S_endo_{Δ19 C1-4}, GST-S_endo_ΔC, GST-S_{endo K1227A}, GST-S_endo_T1220A using 1 µg of each GST fusion protein. 5 µL of cell lysate was used as input control for B. & C. (8% of volume used in each pull down); 10 µL of cell lysate was used as input control for D. (5% of volume used in each pull down). IB: Immunoblot. Results shown are representative of two independent experiments.</p

Effect of wt or FERM ezrin expression on S-mediated cell-cell fusion.

<p>A. Microscopy analysis of syncytia formation induced by activated SARS-CoV S. Vero E6 GFP, Vero E6 GFP-ezrin_wt and Vero E6 GFP-ezrin_FERM cells were overlaid on HeLa cells stably expressing HcRed alone or HcRed and SARS-CoV S. SARS-CoV S was activated or not with trypsin for 15 minutes (+Trypsin). 18 hours later, cells were fixed, nuclei stained with DAPI and analyzed by microscopy for syncytia. Arrows indicate syncytia. B. Quantification of syncytia. For the conditions in which HeLa HcRed Spike cells (+Trypsin) were incubated with Vero E6 GFP or Vero E6 GFP-ezrin_wt or Vero E6 GFP-ezrin_FERM cells, 10 random microscopy fields were analyzed for total number of nuclei (DAPI) and number of nuclei in multi-nucleated cells (DAPI/HcRed/GFP). Results are representative of three independent experiments. ** indicates a value of <i>p</i><0.001 in two-tailed t-tests.</p

Knock down of ezrin by siRNA increases entry of SARS-CoV S pseudotyped particles.

<p>A. Ezrin expression knock down by siRNA. HeLa-F5 cells stably expressing the SARS-CoV receptor ACE2 were transfected twice with ezrin or non-targeting siRNAs and knock down efficiency was estimated by Western blot analysis of ezrin and actin content of cell lysates. B. Luciferase activity fold change analysis. HeLa-F5 cells treated with siRNAs as indicated above were infected with pseudotyped lentiviral particles harboring the VSV G or SARS-CoV S viral envelope glycoproteins. Δenvpp indicates lentiviral particles without any viral surface glycoprotein. The fold change in luciferase activity was calculated using the non-targeting siRNA condition as negative control. Experiments were performed in triplicates and the results of this experiment are representative of at least three independent experiments. ** indicates a value of <i>p</i><0.001 in two-tailed t-tests.</p

Expression of the FERM domain of ezrin increases Vero E6 cell susceptibility to SARS-CoV infection.

<p>A. Flow cytometry analysis (a.) and subcellular localization (b.) of the wt or FERM domain GFP-ezrin in clonal Vero E6 stable cell lines. (b.) Arrows indicate enrichments of wt or FERM ezrin. Scale bars: 20 µm. B. Time course of SARS-CoV replication in Vero E6 stable cell lines expressing wt or FERM GFP-ezrin. Vero E6, Vero E6 GFP-ezrin_wt, Vero E6 GFP-ezrin_FERM were infected with SARS-CoV (strain HK39849) at M.O.I. 5. At 3, 6 and 24 hours post infection, SARS-CoV N RNA levels were measured using qRT-PCR with 18S rRNA normalization. For each condition the average of two measurements of two independent wells was calculated. C. SARS-CoV infection rates in Vero E6, Vero E6 GFP-ezrin_wt and Vero E6 GFP-ezrin_FERM cell lines. Cells were infected with SARS-CoV (strain HK39849) at M.O.I. of 5. 24 h post-infection, cells were immunolabeled for SARS-CoV S. (a.), scale bars: 100 µm. (b.) for each cell line, images of ten random microscopy fields were acquired and analyzed for total number of cells (n; DAPI or Phalloidin AMCA staining) and SARS-CoV S positive cells (TRITC staining) using Imaris 6.3 software. ** indicates a value of <i>p</i><0.001 in two-tailed t-tests.</p

Ezrin accumulates at sites of entry of SARS-CoV S pseudotyped particles.

<p>Vero E6 cells stably expressing RFP-ezrin were inoculated with SARS-CoV S-pseudotyped lentiviral particles harboring a GFP-tagged Vpr protein (SARSpp GFP-Vpr) on ice for 30 minutes. Unbound particles were washed. Internalization of particles was induced by placing the culture dish in a 37°C 5% CO₂ chamber. At 30 minutes post temperature switch (t = 0), cells were analyzed by TIRFM. Time-lapse images were acquired every 3 seconds. The whole cell (first frame) is shown on the left panel. The region shown for time-lapse images is indicated by a square. 3 frames at t = 33, 78 and 108 seconds out of a total of 50 frames are shown and correspond to the times after the start of image acquisitions. The movie of this image sequence is shown as supporting material (Supporting movie S1). Scale bar = 40 µm.</p

The F1 lobe of the ezrin FERM domain binds to the SARS-CoV S endodomain.

<p>A. Table summarizing the yeast two-hybrid screening results for ezrin - S endodomain interaction. B. Schematic representation of the SARS-CoV S endodomain sequence (not drawn to scale). S1: Subunit 1. S2: Subunit 2. TM: transmembrane domain. endo: endodomain. C. Schematic representation of ezrin (not drawn to scale). The bold line represents the common sequence of the 82 cDNAs corresponding to ezrin found in the yeast two-hybrid screening. The domain was named ezrin F1/Spike Binding Domain (SBD). F1, F2, and F3 represent approximate regions corresponding to the three lobes of the FERM domain. T567: threonine 567. D. Representation of the three-dimensional crystal structure of the ezrin FERM domain, showing the three distinct globular lobes F1, F2, and F3. The region in light grey represents amino-acids of the F1/SBD. The FERM domain crystal structure for ezrin (PDB ID: 1NI2) was downloaded from the Protein Data Bank (<a href="http://www.pdb.org" target="_blank">http://www.pdb.org</a>). 3-D rendering of the ezrin FERM domain was performed using MacPyMol software (DeLano Scientific).</p