1. Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity
- Author
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Park, Sang Ho, Siddiqi, Haley, Castro, Daniela V, De Angelis, Anna A, Oom, Aaron L, Stoneham, Charlotte A, Lewinski, Mary K, Clark, Alex E, Croker, Ben A, Carlin, Aaron F, Guatelli, John, and Opella, Stanley J
- Subjects
Biological Sciences ,Bioinformatics and Computational Biology ,Infectious Diseases ,Biodefense ,Vaccine Related ,Prevention ,Lung ,Emerging Infectious Diseases ,Pneumonia ,Underpinning research ,2.2 Factors relating to the physical environment ,Development of treatments and therapeutic interventions ,5.1 Pharmaceuticals ,1.1 Normal biological development and functioning ,Aetiology ,Infection ,Amiloride ,Animals ,Antiviral Agents ,Binding Sites ,COVID-19 ,Chlorocebus aethiops ,Coronavirus Envelope Proteins ,Humans ,Ion Channels ,Nuclear Magnetic Resonance ,Biomolecular ,Protein Binding ,Protein Conformation ,Protein Domains ,SARS-CoV-2 ,Vero Cells ,Virus Assembly ,COVID-19 Drug Treatment ,Microbiology ,Immunology ,Medical Microbiology ,Virology ,Medical microbiology - Abstract
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.
- Published
- 2021