Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs
David M Kern1,2,3, Ben Sorum1,2,3, Sonali S Mali1,2
1Department of Molecular & Cell Biology, University of California Berkeley, Berkeley, CA, USA.
Abstract:
SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.
Insights
The SARS-CoV-2 ORF3a protein functions as an ion channel. Inhibiting this viral protein could lead to new treatments for COVID-19 and other coronavirus diseases.
Area of Science:
- Virology
- Structural Biology
- Molecular Biology
Background:
- The SARS-CoV-2 ORF3a protein is a viral ion channel involved in key pathogenic processes.
- The 3a protein and antibodies against it are present in infected patients.
- Targeting the 3a protein in SARS-CoV-1 reduced viral load and disease severity in animal models.
Purpose of the Study:
- To determine the structure of the SARS-CoV-2 3a protein using cryo-electron microscopy.
- To investigate the ion channel activity of the 3a protein.
- To identify potential therapeutic inhibitors of 3a protein function.
Main Methods:
- Cryo-electron microscopy (cryo-EM) for structure determination.
- Electrophysiology and fluorescent ion imaging in reconstituted liposomes.
- Site-directed mutagenesis to study ion permeability.
Main Results:
- The SARS-CoV-2 3a protein was resolved to 2.1-Å resolution, revealing a novel fold with distinct openings.
- The protein exhibits Ca2+-permeable, nonselective cation channel activity.
- Specific mutations were found to alter ion permeability, and polycationic compounds were identified as inhibitors.
Conclusions:
- The 3a protein's structure and ion channel activity provide insights into its role in viral pathogenesis.
- 3a-like proteins are conserved across various coronaviruses, suggesting broad therapeutic potential.
- Targeting the SARS-CoV-2 3a protein offers a promising strategy for developing treatments against COVID-19 and other coronavirus infections.
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