Poster Presentation 50th Lorne Proteins Conference 2025

Reconstitution of ACE2-S1-RBD interaction on supported lipid bilayer reveals endocytosis-mediated Omicron S1-RBD internalization  (#205)

Angelin Philip 1 , S M Nasir Uddin 1 , Zeyaul Islam 2 , Prasanna R Kolatkar 2 , Kabir H Biswas 1
  1. Hamad Bin Khalifa University, Doha Al Rayyan, DOHA, Qatar
  2. Diabetes Research Center, Qatar Biomedical Research Institute, Doha, Qatar

The SARS-CoV-2 spike protein, through its receptor binding domain (S1-RBD), binds to the ACE2 receptor on host cell membrane leading to viral infection. Additionally, mutations in S1-RBD in SARS-CoV-2 variants have been known to enhance infection, likely due to an increased binding to ACE2. While many reports are available describing SARS-CoV-2 infection mechanism and the impact of mutations in the S1-RBD, there is dearth of studies to understand the initial interaction of the S1-RBD with ACE2 in living cells and on the role of endocytosis and cytoskeleton in the host cell. Here, we reconstituted the interaction between S1-RBD and ACE2 expressing host cells in a hybrid live cell-supported lipid bilayer (SLB) platform and show that cells specifically depleted Omicron S1-RBD-mGLfrom SLB corrals, likely, through endocytosis. Specifically, we prepared either planar or micropatterned SBL substrates and functionalized them with either Wuhan (WT) or Omicron or Revertant S1-RBD-mGL fused with mGreenLantern (mGL) through Ni-NTA-poly-His-tag interaction. Interaction of A549 cells expressing ACE2 resulted in an enrichment of S1-RBD- mGL at cell-SLB interaction. Similar experiments comparing either WT or Omicron or Revertant S1-RBD-mGL on micropatterned SLBs revealed a depletion of the Omicron S1-RBD-mGL, a slight depletion in the Revertant S1-RBD-mGL but not that of WT S1-RBD from SLB corrals, likely through enhanced localization with the endocytic protein EEA1. Indeed, treatment of cells with clathrin-mediated endocytosis inhibitor, pitstop2, but not myosin inhibitor, blebbistatin, resulted in a large decrease in the depletion of Omicron S1-RBD-mGL from the SLB corrals. These suggests a role for endocytosis in the initial interaction of the S1-RBD and the host cell and more importantly, an enhanced interaction of the Omicron S1-RBD-mGL leading to its depletion. However, the slight depletion in the Revertant S1-RBD-mGL was reduced with blebbistatin. Our findings presented here demonstrate, for the first time, the depletion of the protein from membrane surfaces. This underscores the significance of the hybrid live cell SLB platform in studying receptor-ligand complex interactions, the role of cellular processes in viral infection, and the potential impact of mutations in the spike RBD on narrowing or adapting to a specific cellular entry mechanism.

 

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