tetano
Editor, Senior Moderator
PLoS Pathog
. 2021 May 18;17(5):e1009519.
doi: 10.1371/journal.ppat.1009519. Online ahead of print.
Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity
Sang Ho Park[SUP] 1 [/SUP], Haley Siddiqi[SUP] 1 [/SUP], Daniela V Castro[SUP] 1 [/SUP], Anna A De Angelis[SUP] 1 [/SUP], Aaron L Oom[SUP] 2 [/SUP], Charlotte A Stoneham[SUP] 3 [/SUP], Mary K Lewinski[SUP] 3 [/SUP], Alex E Clark[SUP] 3 4 [/SUP], Ben A Croker[SUP] 5 [/SUP], Aaron F Carlin[SUP] 3 [/SUP], John Guatelli[SUP] 3 [/SUP], Stanley J Opella[SUP] 1 [/SUP]
Affiliations
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.
. 2021 May 18;17(5):e1009519.
doi: 10.1371/journal.ppat.1009519. Online ahead of print.
Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity
Sang Ho Park[SUP] 1 [/SUP], Haley Siddiqi[SUP] 1 [/SUP], Daniela V Castro[SUP] 1 [/SUP], Anna A De Angelis[SUP] 1 [/SUP], Aaron L Oom[SUP] 2 [/SUP], Charlotte A Stoneham[SUP] 3 [/SUP], Mary K Lewinski[SUP] 3 [/SUP], Alex E Clark[SUP] 3 4 [/SUP], Ben A Croker[SUP] 5 [/SUP], Aaron F Carlin[SUP] 3 [/SUP], John Guatelli[SUP] 3 [/SUP], Stanley J Opella[SUP] 1 [/SUP]
Affiliations
- PMID: 34003853
- DOI: 10.1371/journal.ppat.1009519
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.