The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a ß-Sheet Bundle in Lipid Bilayers.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a ß-sheet-rich conformation that contains three ß-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third ß-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the ß-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt ß-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the ß-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.

SARS-CoV-2 accessory protein 7b forms homotetramers in detergent.

A global pandemic is underway caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome, like its predecessor SARS-CoV, contains open reading frames that encode accessory proteins involved in virus-host interactions active during infection and which likely contribute to pathogenesis. One of these accessory proteins is 7b, with only 44 (SARS-CoV) and 43 (SARS-CoV-2) residues. It has one predicted transmembrane domain fully conserved, which suggests a functional role, whereas most variability is contained in the predicted cytoplasmic C-terminus. In SARS-CoV, 7b protein is expressed in infected cells, and the transmembrane domain was necessary and sufficient for Golgi localization. Also, anti-p7b antibodies have been found in the sera of SARS-CoV convalescent patients. In the present study, we have investigated the hypothesis that SARS-2 7b protein forms oligomers with ion channel activity. We show that in both SARS viruses 7b is almost completely α-helical and has a single transmembrane domain. In SDS, 7b forms various oligomers, from monomers to tetramers, but only monomers when exposed to reductants. Combination of SDS gel electrophoresis and analytical ultracentrifugation (AUC) in both equilibrium and velocity modes suggests a dimer-tetramer equilibrium, but a monomer-dimer-tetramer equilibrium in the presence of reductant. This data suggests that although disulfide-linked dimers may be present, they are not essential to form tetramers. Inclusion of pentamers or higher oligomers in the SARS-2 7b model were detrimental to fit quality. Preliminary models of this association was generated with AlphaFold2, and two alternative models were exposed to a molecular dynamics simulation in presence of a model lipid membrane. However, neither of the two models provided any evident pathway for ions. To confirm this, SARS-2 p7b was studied using Planar Bilayer Electrophysiology. Addition of p7b to model membranes produced occasional membrane permeabilization, but this was not consistent with bona fide ion channels made of a tetrameric assembly of α-helices.

SARS-CoV 2 accessory protein 7b forms homotetramers in detergent (preprint)

A global pandemic is underway caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 genome, like its predecessor SARS-CoV, contains open reading frames that encode accessory proteins involved in virus-host interactions active during infection and which likely contribute to pathogenesis. One of these accessory proteins is 7b (p7b), with only 44 and 43 residues in SARS-CoV and SAR-CoV-2, respectively. It has one predicted transmembrane domain fully conserved, which suggests a functional role, whereas most variability is contained in the predicted cytoplasmic C-terminus. In SARS-CoV, 7b protein is expressed in infected cells where the transmembrane domain was necessary and sufficient for Golgi localization. Also, anti-p7b antibodies have been found in the sera of SARS-CoV convalescent patients. In the present study, we have investigated the hypothesis that SARS-2 7b protein forms oligomers with ion channel activity. We show that 7b protein is almost completely α-helical in both SARS viruses and has a single transmembrane domain. In SDS, p7b forms various oligomers, from monomers to tetramers, but only monomers when exposed to reductants DTT or TCEP. Combination of SDS gel electrophoresis and and analytical ultracentrifugation (AUC) in both equilibrium and velocity modes suggests a dimer-tetramer equilibrium, and an equilibrium between monomeric, dimeric and tetrameric forms in the presence of reductant. Inclusion of pentamers or higher oligomers in the SARS-2 7b model worsened the fits. Although sensitivity to reductants suggests the involvement of disulfide-linked dimers, the presence of disulfide bonds was not essential to form tetramers. A preliminary model of this association was generated with Alpha-Fold, which was exposed to a molecular dynamics simulation in presence of a model lipid membrane. However, neither of the two models provide any evident pathway for ions. To confirm this, SARS-2 p7b was studied using Planar Bilayer Electrophysiology. Addition of p7b produced occasional membrane permeabilization that is not consistent with bona fide ion channels made of a tetrameric assembly of α-helices.

Transport mechanisms of SARS-CoV-E viroporin in calcium solutions: Lipid-dependent Anomalous Mole Fraction Effect and regulation of pore conductance.

The envelope protein E of the SARS-CoV coronavirus is an archetype of viroporin. It is a small hydrophobic protein displaying ion channel activity that has proven highly relevant in virus-host interaction and virulence. Ion transport through E channel was shown to alter Ca2+ homeostasis in the cell and trigger inflammation processes. Here, we study transport properties of the E viroporin in mixed solutions of potassium and calcium chloride that contain a fixed total concentration (mole fraction experiments). The channel is reconstituted in planar membranes of different lipid compositions, including a lipid mixture that mimics the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane where the virus localizes within the cell. We find that the E ion conductance changes non-monotonically with the total ionic concentration displaying an Anomalous Mole Fraction Effect (AMFE) only when charged lipids are present in the membrane. We also observe that E channel insertion in ERGIC-mimic membranes - including lipid with intrinsic negative curvature - enhances ion permeation at physiological concentrations of pure CaCl2 or KCl solutions, with a preferential transport of Ca2+ in mixed KCl-CaCl2 solutions. Altogether, our findings demonstrate that the presence of calcium modulates the transport properties of the E channel by interacting preferentially with charged lipids through different mechanisms including direct Coulombic interactions and possibly inducing changes in membrane morphology.