Irreversible Inactivation of SARS-CoV-2 by Lectin Engagement with Two Glycan Clusters on the Spike Protein

Abstract

Host cell infection by SARS-CoV-2, similar to that by HIV-1, is driven by a conformationally metastable and highly glycosylated surface entry protein complex, and infection by these viruses has been shown to be inhibited by the mannose-specific lectins cyanovirin-N (CV-N) and griffithsin (GRFT). We discovered in this study that CV-N not only inhibits SARS-CoV-2 infection but also leads to irreversibly inactivated pseudovirus particles. The irreversibility effect was revealed by the observation that pseudoviruses first treated with CV-N and then washed to remove all soluble lectin did not recover infectivity. The infection inhibition of SARS-CoV-2 pseudovirus mutants with single-site glycan mutations in spike suggested that two glycan clusters in S1 are important for both CV-N and GRFT inhibition: one cluster associated with the RBD (receptor binding domain) and the second with the S1/S2 cleavage site. We observed lectin antiviral effects with several SARS-CoV-2 pseudovirus variants, including the recently emerged omicron, as well as a fully infectious coronavirus, therein reflecting the breadth of lectin antiviral function and the potential for pan-coronavirus inactivation. Mechanistically, observations made in this work indicate that multivalent lectin interaction with S1 glycans is likely a driver of the lectin infection inhibition and irreversible inactivation effect and suggest the possibility that lectin inactivation is caused by an irreversible conformational effect on spike. Overall, lectins' irreversible inactivation of SARS-CoV-2, taken with their breadth of function, reflects the therapeutic potential of multivalent lectins targeting the vulnerable metastable spike before host cell encounter.

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

“…S17). Interestingly, site-directed mutagenesis demonstrated that the N657Q/D point mutation increased infectivity for reasons not yet clarified (Nangarlia et al, 2023; Yang et al, 2022). Visual inspection and H-bond analysis show that the N657 glycan gains interactions with D843 solely upon ACE2 binding (Fig.…”

“…4B, Fig. S10, 12,13). Similarly, in the third intermediate state, the longer dissociation time in the presence of GAGs is due to stronger interactions established between the ACE2 N90 glycan and the spike RBDs, which result in a narrower crevice where the N-glycan is inserted (Fig.…”

“…66 N-glycans are covalently attached to the protein surface of the trimeric spike ectodomain (Grant et al, 2020; Watanabe et al, 2020; Xie & Butler, 2023; Zhao et al, 2020). Beyond generally shielding the protein surface, these N-glycans have been found to control spike activation, modulate the protein:protein interactions, and sterically mask functional epitopes of the viral protein (Casalino et al, 2020; Harbison et al, 2022; Mori et al, 2021; Nangarlia et al, 2023; Newby et al, 2023; Sztain et al, 2021; Q. Yang et al, 2020).…”

“…66 N-glycans are covalently attached to the protein surface of the trimeric spike ectodomain [5][6][7][8] . Beyond generally shielding the protein surface, these N-glycans have been found to control spike activation, modulate the protein:protein interactions, and sterically mask functional epitopes of the viral protein [9][10][11][12][13][14][15][16] . The prefusion spike has been shown to adopt inactive closed and active open conformations 1 .…”

The Effect of Select SARS-CoV-2 N-Linked Glycan and Variant of Concern Spike Protein Mutations on C-Type Lectin-Receptor-Mediated Infection

“…There are already prophylactic strategies against other viruses that leverage binding to glycans on the viral spike protein: several anti-HIV antibodies bind to the glycan shield rather than amino acid epitopes, and other globular-lectin-protein-based inhibitors such as griffithsin exhibit broad inhibitory activity against a variety of viruses [ 54 , 111 , 112 , 113 ]. Recent literature has shown considerable success when griffithsin treatment was combined with a therapeutic that targeted a different epitope on the SARS-CoV-2 spike—a designed viral fusion inhibitor called EK1, sulfated polysaccharides called carrageenan, or another glycan-binding inhibitor called cyanovirin [ 114 , 115 , 116 , 117 ]. These results hint that if new inhibitors could be designed against unique SARS-CoV-2 epitopes (such as the N17/61/74 or N122/145/234 glycan clusters), they could be combined with other existing COVID-19 therapeutics to create a more potent prophylactic treatment.…”