Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity

PLoS Pathog. 2021 May 18;17(5):e1009519. doi: 10.1371/journal.ppat.1009519. eCollection 2021 May.


SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amiloride / pharmacokinetics
  • Amiloride / pharmacology*
  • Animals
  • Antiviral Agents / pharmacology
  • Binding Sites / drug effects
  • COVID-19 / drug therapy*
  • COVID-19 / virology
  • Chlorocebus aethiops
  • Coronavirus Envelope Proteins / chemistry
  • Coronavirus Envelope Proteins / metabolism*
  • Humans
  • Ion Channels / metabolism
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Binding / drug effects
  • Protein Conformation / drug effects
  • Protein Domains
  • SARS-CoV-2 / drug effects*
  • SARS-CoV-2 / metabolism*
  • Vero Cells
  • Virus Assembly / drug effects


  • Antiviral Agents
  • Coronavirus Envelope Proteins
  • Ion Channels
  • envelope protein, SARS-CoV-2
  • Amiloride