17 research outputs found
Rôles des lectines de type C L-SIGN et DC-SIGN dans l'infection par le virus de l'hépatite C
No full textPARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF
Motyw władzy w dramacie Bum Mariusa von Mayenburga
No full textPower is the main theme of the play Bang, which polish premiered in 2019 and took place at the New Theater of Kazimierz Dejmek in Łódź. The play was directed based on the drama of the same title, written by the contemporary German author and playwright Marius von Mayenburg. The aim of the article is to analyze and describe the means by which the viewer has the opportunity to understand why the theme of power is highlighted against the background of others presented in the play and to answer the question of what is the reason for the main character Ralf Bang’s desire to exercise power even before his own birth
Ezrin interacts with the SARS coronavirus spike protein and restrains infection at the entry stage
No full text© 2012 Millet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.This work was supported by the Research Grant Council of Hong Kong (RGC#760208)and the RESPARI project of the International Network of Pasteur Institutes
Ezrin accumulates at sites of entry of SARS-CoV S pseudotyped particles.
No full text<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% CO<sub>2</sub> 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
<i>In vitro</i> confirmation of the interaction between SARS-CoV S endodomain and ezrin.
No full text<p>A. The endodomain of S pulls down ezrin from HeLa cell lysate. Cell lysate was incubated with Glutathione-Sepharose beads either uncoupled (lane 2) or coupled with the fusion protein GST-FERM (positive control, lane 3) or increasing amounts of GST-S<sub>endo</sub> (lanes 4–6). B. The endodomain of S pulls down ezrin from Vero E6 cell lysate. Similarly, cell lysate was incubated with Glutathione-Sepharose beads bound either to GST fused to S<sub>endo</sub> in absence (lanes 2–3) or presence (lanes 4–5) of rabbit serum that recognizes the endodomain of S, or to GST alone (lane 6). For both A. and B. 10 µL of cell lysate was used as input control which represents 1.6% and 4% of the volume used for the pull down for A. and B., respectively. IB: Immunoblot. Results shown are representative of two independent experiments.</p
Characterization of interactions determinants of S endodomain binding to ezrin.
No full text<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<sub>endo </sub><sub>wt</sub> (B. & C.), GST-S<sub>endo Δ8</sub>, and GST-S<sub>endo Δ19</sub>(B.), GST-S<sub>endo C1</sub>, GST-S<sub>endo C2</sub>, GST-S<sub>endo C3</sub>, GST-S<sub>endo C4</sub>, and GST-S<sub>endo C1-4</sub> (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<sub>endo wt</sub>, GST-S<sub>endo </sub><sub>Δ8 C1-4</sub>, and GST-S<sub>endo </sub><sub>Δ19 C1-4</sub>, GST-S<sub>endo </sub><sub>ΔC</sub>, GST-S<sub>endo K1227A</sub>, GST-S<sub>endo </sub><sub>T1220A</sub> 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
Knock down of ezrin by siRNA increases entry of SARS-CoV S pseudotyped particles.
No full text<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.
No full text<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<sub>wt</sub>, Vero E6 GFP-ezrin<sub>FERM</sub> 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<sub>wt</sub> and Vero E6 GFP-ezrin<sub>FERM</sub> 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
Effect of wt or FERM ezrin expression on S-mediated cell-cell fusion.
No full text<p>A. Microscopy analysis of syncytia formation induced by activated SARS-CoV S. Vero E6 GFP, Vero E6 GFP-ezrin<sub>wt</sub> and Vero E6 GFP-ezrin<sub>FERM</sub> 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<sub>wt</sub> or Vero E6 GFP-ezrin<sub>FERM</sub> 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
