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

Abstract 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.