Ezrin interacts with the SARS coronavirus spike protein and restrains infection at the entry stage

© 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

Wandering ideal point models for single or multi-attribute ranking data: a Bayesian approach

published_or_final_versionabstracttocStatistics and Actuarial ScienceMasterMaster of Philosoph

Transmission potential of influenza in respiratory droplets

Influenza virus is a respiratory infection of public health importance, because it is associated with a substantial burden of morbidity and mortality in Hong Kong and elsewhere. The traditional paradigm suggests that influenza virus transmits between humans primarily by respiratory droplets and secondarily by contact, while there is controversy over the role of aerosol transmission. Interventions to control influenza epidemics often require identification of infected persons, which is challenging without laboratory testing, while asymptomatic infections may have a role in community spread. I conducted a series of studies on the potential contribution of aerosols to the transmission of influenza, the efficacy of surgical face masks against aerosols, the potential for a new rapid test to be used for the identification of cases of influenza virus infection, and the potential importance of asymptomatic infections in the spread of influenza epidemics. In one study, I used a Gesundheit-II (G-II) device to collect exhaled breath particles from outpatients in two size fractions, ≥5μm (coarse) and <5μm (fine) and maintained viability of any viruses collected in the fine fraction. In a second study, I used a cyclone NIOSH air sampler to detect influenza virus in fine particles in patient rooms. In the third study, I used data from three separate studies of outpatients to characterize the clinical diagnostic performance of the Sofia Influenza A+B Fluorescent Immunoassay. Finally, I conducted a systematic review and meta-analysis of published estimates of the proportion of influenza virus infections that are asymptomatic. In the first study, I showed that influenza A virus, including A(H1N1)pdm09 and A(H3N2), were found in both fine and coarse fractions of exhaled breath particles. I also showed that wearing a surgical face mask led to a significant and substantial reduction in influenza A virus shedding in the coarse fraction of exhaled breath. In the second study I detected influenza virus RNA in aerosols at low concentrations half of the time in patient rooms with patients with confirmed influenza virus infection. I estimated that the sensitivity and specificity of the Sofia rapid test for detection of influenza A and B were 77% (95% CI 71-82%) and 91% (95% CI 89-92%) respectively. In the meta-analysis I found that estimates of the asymptomatic fraction were affected by study design, with estimates from outbreak investigations having a pooled mean of 16% (95% CI: 13%, 19%), while estimates from cohort studies adjusting for incidence of non-influenza illnesses fell in the range 65%-85%. Influenza virus could be detected in fine particles in the exhaled breath of outpatients with influenza, and in the rooms of inpatients with confirmed influenza, supporting a role of aerosol transmission in the spread of influenza in the community. The Sofia rapid test had higher sensitivity than other point-of-care tests, but poorer specificity leading to a low positive predictive value outside of peaks in influenza activity. Uncertainty remains about the frequency of asymptomatic infections in the community which depends on how infections are identified, and their importance in transmission.published_or_final_versionPublic HealthDoctoralDoctor of Philosoph

Mechanism of antibody-dependent enhancement in severe acute respiratory syndrome coronavirus infection

Severe lymphopenia is a clinical feature of Severe Acute Respiratory Syndrome (SARS) patients. However, lymphocytes do not express receptor for SARS-CoV, neither the widely accepted viral receptor angiotensin converting enzyme 2 (ACE2) nor the putative receptors Dendritic Cell- and Liver/lymph-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN and L-SIGN). Our group previously showed in vitro that, SARS-CoV Spike pseudotyped particles (SARSCoVpp) could infect human B cells only when inoculated in presence of anti-SARSCoV Spike immune serum. Such observations raised concerns about the possible occurrence of antibody-dependent enhancement (ADE) of infection, a phenomenon during which a virus bounded by antibodies could gain entry into cells through mechanisms involving complement receptors or Fc receptors. Recently, we have demonstrated the participation of the human Fc gamma receptor II (hFcγRII) molecules in granting SARS-CoV an opportunity to infect human immune cells. The aim of this study was to decipher the molecular mechanism leading to antibodymediated, FcγRII-dependent infection of immune cells by SARS-CoV. By using transduction experiment, I highlighted that different members of the hFcγRII family (namely hFcγRIIA, hFcγRIIB1 and hFcγRIIB2) could confer susceptibility to ADE of SARS-CoVpp infection. I further demonstrated that purified anti-viral immunoglobulin G, but not other soluble factor(s) from heat-inactivated immune serum, was the determinant for occurrence of ADE infection. Additionally, with the development of a cell-cell fusion assay, I illustrated that in contrast to the ACE2- dependent pathway, ADE infection did not occur at the plasma membrane, but rather require internalization of virus/antibodies immune complexes by the target cells. In line with this hypothesis, my results using a panel of FcγRII-expressing mutants demonstrated that binding of immune complexes to cell surface FcγRII was a prerequisite but was not sufficient to trigger ADE infection. In these experiments, only FcγRII signaling-competent constructions conferred susceptibility to ADE of SARS-CoVpp infection. Altogether my results point toward a role of the anti-SARS-CoV Spike IgG in vitro in granting SARS-CoV an opportunity to infect cells bearing signaling-competent FcγRII receptors. If further confirmed, such observations could have implications for understanding SARS-CoV tropism and SARS pathogenesis, as well as warrant for careful design of SARS vaccines and immunotherapy based on anti-viral antibodies.published_or_final_versionMicrobiologyMasterMaster of Philosoph

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

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