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

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 {approx} 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. Author SummaryThe novel coronavirus SARS-CoV-2, the causative agent of the world-wide pandemic of COVID-19, has become one of the greatest threats to human health. While rapid progress has been made in the development of vaccines, drug discovery has lagged, partly due to the lack of atomic-resolution structures of the free and drug-bound forms of the viral proteins. The SARS-CoV-2 envelope (E) protein, with its multiple activities that contribute to viral replication, is widely regarded as a potential target for COVID-19 treatment. As structural information is essential for drug discovery, we established an efficient sample preparation system for biochemical and structural studies of intact full-length SARS-CoV-2 E protein and characterized its structure and dynamics. We also characterized the interactions of amilorides with specific E protein residues and correlated this with their antiviral activity during viral replication. The binding affinity of the amilorides to E protein correlated with their antiviral potency, suggesting that E protein is indeed the likely target of their antiviral activity. We found that residue asparagine15 plays an important role in maintaining the conformation of the amiloride binding site, providing molecular guidance for the design of inhibitors targeting E protein.

Functional Landscape of SARS-CoV-2 Cellular Restriction (preprint)

A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.