Targeting conserved N-glycosylation blocks SARS-CoV-2 variant infection in vitro

Abstract

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

Inhibition of Glycolysis Impairs Retinoic Acid-Inducible Gene I–Mediated Antiviral Responses in Primary Human Dendritic Cells

“…As observed with DENV infection, a strong reduction in hCoV-229E vRNA expression (≈90%) was exerted by M8 and was maintained in the presence of 2-DG but not 3-PO (≈50% recovery) ( Figure 5E ). However, 2-DG reduced the levels of 229E vRNA levels even in the absence of M8, consistent with the hypothesis that block of N-glycosylation affects the infectious process of enveloped viruses, as observed also with other coronaviruses ( Codo et al., 2020 ; Huang et al., 2021 ). Finally, to verify whether the antiviral effect of 2-DG on both DENV and hCoV-229E replication was related to the block of N-glycosylation, we infected MoDCs with the non-enveloped virus Coxsackie B5 (CV-B5).…”

Principles of SARS-CoV-2 glycosylation

“…For example, P681H and P681R mutation found in highly transmissible alpha and delta variants respectively, decreases O-glycosylation which potentially increases furin cleavage and may influence viral infectivity [ 53 ]. Together these results have highlighted the importance of S protein glycosylation on viral entry and abrogation of these glycans using inhibitors provides insight into intervention strategies to target the SARS-CoV-2 infection [ 58 , 59 ].…”

Assessment of COVID-19 vaccination effectiveness

“…Zmiana aminokwasów ułatwia bowiem zmiany glikozylacyjne. Obserwowano to stopniowo w czasie pojawiania się kolejnych wariantów wirusa SARS-CoV-2 [10], przy czym wirus, unikając odpowiedzi humoralnej, może zmienić częściowo swój tropizm, tj. zdolność infekowania niektórych komórek.…”

“…COVID- waż wirusy, takie jak SARS-CoV-2, przez większość czasu umiejscowione są, w przeciwieństwie do niektórych bakterii, wewnątrz komórek, czyli tam, gdzie przeciwciała nie docierają. Ponadto limfocyty cytotoksyczne są w zasadzie niewrażliwe lub w bardzo małym stopniu wrażliwe na glikozylację, gdyż proces prezentowania białek wirusa znajdującego się wewnątrz komórki ogranicza znaczenie glikozylacji, chociaż nie zawsze ją całkowicie eliminuje [10]. W badaniach dotyczących limfocytów cytotoksycznych wykazano, że pojawiają się one zarówno po przejściu infekcji, jak i po szczepieniu [13,14].…”