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Supplementary Material for: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds with spike protein and inhibits the entry of SARS-CoV-2 into host cells

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posted on 2024-02-07, 11:18 authored by Dilawari R., Chaubey G.K., Modanwal R., Dhiman A., Talukdar S., Kumar A., Raje C.I., Raje M.
Introduction: Coronavirus disease 2019 (COVID-19) caused by coronavirus-2 (SARS-CoV-2) has emerged as an aggressive viral pandemic. Health care providers confront a challenging task for rapid development of effective strategies to combat this and its long term after effects. Virus entry into host cells involves interaction between receptor-binding domain (RBD) of Spike (S) protein S1 subunit with angiotensin converting enzyme (ACE) present on host cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme involved in cellular glycolytic energy metabolism and micronutrient homeostasis. It is deployed in various cellular compartments and the extra cellular milieu. Though it is known to moonlight as a component of mammalian innate immune defense machinery, till date its role in viral restriction remains unknown. Method: Recombinant S protein, the receptor binding domain (RBD) and human GAPDH protein were used for solid phase binding assays and Biolayer interferometry (BLI). Pseudo virus particles expressing four different strain variants of S protein all harboring ZsGreen gene as marker of infection were used for Flow cytometry-based infectivity assays. Results: Pseudo-virus entry into target cells in culture was significantly inhibited by addition of human GAPDH into the extracellular medium. Binding assays demonstrated that human GAPDH binds to S protein and RBD domain of SARS-CoV-2 with nano molar affinity. Conclusions: Our investigations suggest that this interaction of GAPDH interferes in the viral docking with hACE2 receptors, thereby affecting viral ingress into mammalian cells.


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