Background: Despite clinical success with anti-spike vaccines, the effectiveness of neutralizing antibodies and vaccines has been compromised by rapidly spreading SARS-CoV-2 variants. Viruses can hijack the glycosylation machinery of host cells to shield themselves from the host's immune response and attenuate antibody efficiency. However, it remains unclear if targeting glycosylation on viral spike protein can impair infectivity of SARS-CoV-2 and its variants. Methods: We adopted flow cytometry, ELISA, and BioLayer interferometry approaches to assess binding of glycosylated or deglycosylated spike with ACE2. Viral entry was determined by luciferase, immunoblotting, and immunofluorescence assays. Genome-wide association study (GWAS) revealed a significant relationship between STT3A and COVID-19 severity. NF-kB/STT3A-regulated N-glycosylation was investigated by gene knockdown, chromatin immunoprecipitation, and promoter assay. We developed an antibody-drug conjugate (ADC) that couples non-neutralization anti-spike antibody with NGI-1 (4G10-ADC) to specifically target SARS-CoV-2-infected cells. Findings: The receptor binding domain and three distinct SARS-CoV-2 surface N-glycosylation sites among 57,311 spike proteins retrieved from the NCBI-Virus-database are highly evolutionarily conserved (99.67%) and are involved in ACE2 interaction. STT3A is a key glycosyltransferase catalyzing spike glycosylation and is positively correlated with COVID-19 severity. We found that inhibiting STT3A using N-linked glycosylation inhibitor-1 (NGI-1) impaired SARS-CoV-2 infectivity and that of its variants [Alpha (B.1.1.7) and Beta (B.1.351)]. Most importantly, 4G10-ADC enters SARS-CoV-2-infected cells and NGI-1 is subsequently released to deglycosylate spike protein, thereby reinforcing the neutralizing abilities of antibodies, vaccines, or convalescent sera and reducing SARS-CoV-2 variant infectivity. Interpretation: Our results indicate that targeting evolutionarily-conserved STT3A-mediated glycosylation via an ADC can exert profound impacts on SARS-CoV-2 variant infectivity. Thus, we have identified a novel deglycosylation method suitable for eradicating SARS-CoV-2 variant infection in vitro. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section
Human tumor necrosis factor receptor associated factor (TRAF)‐interacting protein, with a forkhead‐associated domain (TIFA), is a key regulator of NF‐κB activation. It also plays a key role in the activation of innate immunity in response to bacterial infection, through heptose 1,7‐bisphosphate (HBP); a metabolite of lipopolysaccharide (LPS). However, the mechanism of TIFA function is largely unexplored, except for the suggestion of interaction with TRAF6. Herein, we provide evidence for direct binding, albeit weak, between TIFA and the TRAF domain of TRAF6, and it is shown that the binding is enhanced for a rationally designed double mutant, TIFA S174Q/M179D. Enhanced binding was also demonstrated for endogenous full‐length TRAF6. Furthermore, the structures of the TRAF domain complexes with the consensus TRAF‐binding peptides from the C terminus of wild‐type and S174Q/M179D mutant TIFA, showing salt‐bridge formation between residues 177–181 of TIFA and the binding pocket residues of the TRAF domain, were solved. Taken together, the results provide direct evidence and a structural basis for the TIFA–TRAF6 interaction, and show how this important biological function can be modulated.
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