Citation
- Authors: Liu J. et al.
- Year: 2022
- Journal: J Biol Chem
- Applications: in vitro / DNA / jetOPTIMUS
- Cell types:
- Name: CHO-K1
Description: Chinese hamster ovary cells - Name: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
- Name: CHO-K1
Method
SARS-CoV-2 RBD into HEK-293T:
SARS-CoV-2 RBD cDNA (T333-K529) was cloned into PHL-mMBP-10 vector (addgene #72348). MBP tagged RBD protein was produced from HEK293T cells. The cells were seeded in DMEM supplemented with 10% FBS for overnight. When cells grew to 70-80% confluency, plasmid DNA was transfected using JetOPTIMUS transfection reagent (Polyplus). After 4 hours, medium was replaced by fresh DMEM without FBS. Conditioned medium containing secreted mMBP-RBD was harvested three days after transfection.
hAce2 into CHO-K1:
Briefly, 1.5x105 CHO-K1 cells (ATCC® CCL-61) were seeded on a microscope coverslips (d=18mm, Fisherbrand) within 12-well plate with 1ml complete medium (DMEM:F12 supplemented with 10% FBS). Cells were incubated under normal growth conditions (37 °C and 5% CO2) for overnight.
hAce2 (addgene #1786) was transfected using JetOPTIMUS transfection reagent (Polyplus) when overnight cell culture grew up to 50-70% confluency. Transfected cells were maintained for another 24 hours. Then medium was removed, cells were washed once with PBS, and replaced with 0.5mL fresh medium.
Abstract
Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is broadly accepted that SARS-CoV-2 utilizes its spike protein to recognize the extracellular domain of angiotensin-converting enzyme 2 (ACE2) to enter cells for viral infection. However, other mechanisms of SARS-CoV-2 cell entry may occur. We show quantitatively that the SARS-CoV-2 spike protein also binds to the extracellular domain of broadly expressed integrin α5β1 with an affinity comparable to that of SARS-CoV-2 binding to ACE2. More importantly, we provide direct evidence that such binding promotes the internalization of SARS-CoV-2 into non-ACE2 cells in a manner critically dependent upon the activation of the integrin. Our data demonstrate an alternative pathway for the cell entry of SARS-CoV-2, suggesting that upon initial ACE2-mediated invasion of the virus in the respiratory system, which is known to trigger an immune response and secretion of cytokines to activate integrin, the integrin-mediated cell invasion of SARS-CoV-2 into the respiratory system and other organs becomes effective, thereby promoting further infection and progression of COVID-19.
