The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione

• Published in: Biochemical and biophysical research communications Vol. 597; pp. 30 - 36
• Main Authors: Murae, Mana, Shimizu, Yoshimi, Yamamoto, Yuichiro, Kobayashi, Asuka, Houri, Masumi, Inoue, Tetsuya, Irie, Takuya, Gemba, Ryutaro, Kondo, Yosuke, Nakano, Yoshio, Miyazaki, Satoru, Yamada, Daisuke, Saitoh, Akiyoshi, Ishii, Isao, Onodera, Taishi, Takahashi, Yoshimasa, Wakita, Takaji, Fukasawa, Masayoshi, Noguchi, Kohji
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.2022
• Subjects: Cysteine; SARS-CoV-2; Spike; Spike; SARS-CoV-2; Cysteine
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2022.01.106

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. •SARS-CoV-2 spike protein plays important roles in the viral entry process.•In vitro functions by variant spike receptor binding domain (RBD) and ACE2 were inhibited by NAC and GSH.•The C488A mutation impaired pseudotyped virus infection, syncytium formation, and cell-cell fusion by SARS-CoV-2 spike expression.