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

PLoS One. 2012;7(11):e49566. doi: 10.1371/journal.pone.0049566. Epub 2012 Nov 21.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cell Membrane / metabolism
  • Cytoskeletal Proteins / chemistry*
  • Cytosol / metabolism
  • Gene Library
  • Glutathione Transferase / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / metabolism
  • Molecular Sequence Data
  • Multigene Family
  • Mutation
  • Protein Binding
  • Protein Structure, Tertiary
  • SARS Virus / metabolism*
  • Sequence Homology, Amino Acid
  • Spike Glycoprotein, Coronavirus
  • Two-Hybrid System Techniques
  • Vero Cells
  • Viral Envelope Proteins / chemistry*
  • Viral Envelope Proteins / metabolism

Substances

  • Cytoskeletal Proteins
  • Membrane Glycoproteins
  • Spike Glycoprotein, Coronavirus
  • Viral Envelope Proteins
  • ezrin
  • spike glycoprotein, SARS-CoV
  • Glutathione Transferase

Grant support

This work was supported by the Research Grant Council of Hong Kong (RGC#760208); http://www.ugc.edu.hk/eng/rgc/index.htm. the RESPARI project of the International Network of Pasteur Institutes; http://www.hkupasteur.hku.hk/respari/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.