Citation

  • Authors: Murae M. et al.
  • Year: 2022
  • Journal: Biochem Biophys Res Commun 597 30-36
  • Applications: in vitro / DNA / PEIpro
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

For retrovirus-based pseudotyped virus production, 293T cells were co-transfected with spike-expressing plasmids containing the phCMV-Gag-Pol 5349 and reporter pTG-Luc126 plasmids using the PEIpro® transfection reagent (Polyplus Transfection). Briefly, 2 × 106 293T cells were seeded in a T-25 flask one day before transfection, and the next day, the cells were co-transfected following the manufacturer's instructions. The next day, the growth medium was added to the flask for an additional 2 days of culture. The cell supernatant containing pseudotyped virus was collected, filtered through a 0.45 μm filter, and aliquoted to be stored at −80 °C.

Abstract

Viral spike proteins play important roles in the viral entry process, facilitating attachment to cellular receptors and fusion of the viral envelope with the cell membrane. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor angiotensin converting enzyme-2 (ACE2) via its receptor-binding domain (RBD). The cysteine residue at position 488, consisting of a disulfide bridge with cysteine 480 is located in an important structural loop at ACE2-binding surface of RBD, and is highly conserved among SARS-related coronaviruses. We showed that the substitution of Cys-488 with alanine impaired pseudotyped SARS-CoV-2 infection, syncytium formation, and cell-cell fusion triggered by SARS-CoV-2 spike expression. Consistently, in vitro binding of RBD and ACE2, spike-mediated cell-cell fusion, and pseudotyped viral infection of VeroE6/TMPRSS2 cells were inhibited by the thiol-reactive compounds N-acetylcysteine (NAC) and a reduced form of glutathione (GSH). Furthermore, we demonstrated that the activity of variant spikes from the SARS-CoV-2 alpha and delta strains were also suppressed by NAC and GSH. Taken together, these data indicate that Cys-488 in spike RBD is required for SARS-CoV-2 spike functions and infectivity, and could be a target of anti-SARS-CoV-2 therapeutics.

Pubmed