It is widely accepted that the SARS-CoV-2 betacoronavirus infects humans through binding the human Angiotensin Receptor 2 (ACE2) that lines the nasal cavity and lungs, followed by import into a cell utilizing the Transmembrane Protease, Serine 2 (TMPRSS2) cofactor. ACE2 binding is mediated by an approximately 200-residue portion of the SARS-CoV-2 extracellular spike protein, the receptor binding domain (RBD). Robust interactions are shown using a novel cell-based assay between an RBD membrane tethered-GFP fusion protein and the membrane bound ACE2-Cherry fusion protein. Several observations were not predicted including, quick and sustained interactions leading to internalization of RBD fusion protein into the ACE2 cells and rapid downregulation of the ACE2-Cherry fluorescence. Targeted mutation in the RBD disulfide Loop 4 led to a loss of internalization for several variants tested. However, a secreted RBD did not cause ACE2 downregulation of ACE2-Cherry fluorescence. Thus, the membrane associated form of RBD found on the viral coat may have long-term system wide consequences on ACE2 expressing cells.
Keywords: ACE2 downregulation; NL63; Omicron variant; RBD; SARS-CoV-2; Spike protein; Xbb.1.5 variant; hACE2.