Glyceraldehyde-3-Phosphate Dehydrogenase Binds with Spike Protein and Inhibits the Entry of SARS-CoV-2 into Host Cells

Abstract

Introduction: Coronavirus disease 2019 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 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 RBD, and human GAPDH protein were used for solid phase binding assays and biolayer interferometry. Pseudovirus 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: Pseudovirus 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 of SARS-CoV-2 with nanomolar 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.

Keywords: ACE2; GAPDH; Inhibition; Innate immune defense; Severe acute respiratory syndrome coronavirus-2.

Fig. 1.

a–d Human GAPDH binds with S protein and its RBD peptide segment. A solid phase binding assay was performed using MaxiSorp Nunc plates coated with 100 ng of either S protein (a) or RBD fragment (b). Subsequently, wells were incubated with increasing concentrations of recombinant human GAPDH. After extensive washing, the captured GAPDH was detected with monoclonal anti-GAPDH. Assay was performed multiple times in triplicates. Error bars indicate the standard deviation from triplicate values for each concentration tested. Affinity of interaction was determined by BLI. BLI sensorgrams indicate the binding of recombinant GAPDH with full-length S protein (c) and RBD (d). Data are plotted as response shift in nm versus time and the global equilibrium dissociation constant values (KD) were calculated. e GAPDH from diverse organisms is capable of inhibiting pseudoviral infectivity. Transduction of Vero cells by Wuhan strain S protein pseudovirus is inhibited by GAPDH from evolutionarily diverse sources in human infectivity assay using GAPDH from three organisms, each was tested at different concentrations. BSA was used as a non-specific protein control. All experiments were repeated three times, **p < 0.01. f Human GAPDH can inhibit infection by pseudovirus-expressing spike protein of different COVID-19 variants. Transduction of Vero cells by pseudoviral particles expressing S protein from four different variants is significantly inhibited by human GAPDH, ***p < 0.001, *p < 0.05, n = 3. g, h GAPDH inhibits infection of Vero E6 cells by SARS-CoV-2 virus. When Vero E6 cells were infected with SARS-CoV-2 viral particles in presence of human GAPDH, there was inhibition of viral RNA in supernatant of Vero E6. Viral load was evaluated by RT-PCR of SARS-CoV-2 E gene using total cellular cDNA, *p < 0.05, n = 3 (g) and Western blot of cell lysate to evaluate expression of S protein (h). i, j GAPDH can inhibit SARS-CoV-2 virus infection of Vero E6 host cells in culture up to 12 h post-application. Vero E6 cells were pre-incubated with 100 μg/mL GAPDH or BSA for 12 h at which time cells were rinsed and infected with either pseudoviral particles (i) or SARS-CoV-2 virus (j). After 72 h, the pseudovirus infected cells were evaluated for GFP expression and the % infectivity determined as compared to cells where GAPDH pre-incubation was omitted. In comparison to cells that had been left untreated or pre-treated with BSA, the GAPDH coated cells demonstrate a significantly lower infectivity, ns, not significant, ***p < 0.001, n = 3. SARS-CoV-2 virus infected cells were maintained in culture for 48 h at which time the cellular viral RNA expression was evaluated by RT-PCR for CoV E gene expression. In comparison to control cells, the cells pre-incubated with GAPDH demonstrate significantly lower viral load. ns, not significant, ***p < 0.001, n = 3.