Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

Abstract

BackgroundEntry 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 FindingsWe 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/SignificanceEzrin 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.

Anti-SARS coronavirus agents: a patent review (2008 – present)

“…A WO patent [78] and a Canadian patent [79] as well as their paper [80] published by inventors from Hong-Kong Pasteur Research Center, China, relates to the identification and/ or use of anti-CoV molecules that inhibit or disrupt viral protein-cellular protein interactions, important or essential in the viral life cycle of a CoV, for example, a method for identifying inhibitors of early stages of SARS-CoV replication cycle by using lentiviral Spike pseudotyped particles. Using a yeast two-hybrid screening strategy, the inventors have identified cellular factors that interact with the SARS-CoV's E, S and M proteins, in particular, ezrin (ERM protein) and PALS1 (PDZ-domain containing protein) for interaction with S and E proteins, respectively.…”

Coronaviruses

“…In particular, this report identifi ed nsp1 interaction with several members of immunophilin and calcipressin families, which led to demonstrate SARS sensitivity to cyclosporin A. More recently, another Y2H screen performed with the C-terminal domain of the spike glycoprotein (S) identifi ed Ezrin as a binding partner and a restriction factor of SARS-CoV [ 24 ]. Since 2005, a technological platform is up and running in our laboratory at Institut Pasteur, and dedicated to virus-host interaction mapping for a large panel of RNA viruses.…”

A Field-Proven Yeast Two-Hybrid Protocol Used to Identify Coronavirus–Host Protein–Protein Interactions

“…In particular, this report identifi ed nsp1 interaction with several members of immunophilin and calcipressin families, which led to demonstrate SARS sensitivity to cyclosporin A. More recently, another Y2H screen performed with the C-terminal domain of the spike glycoprotein (S) identifi ed Ezrin as a binding partner and a restriction factor of SARS-CoV [ 24 ].…”