Investigations into the Impact of Glycosylation on Coronavirus Entry and on Toll-like Receptor 4 Activation

Abstract

The COVID-19 pandemic has highlighted our vulnerability to emerging coronaviruses (CoVs). To identify therapeutic targets for future emerging CoVs, we characterized conserved glycan-dependent mechanisms of CoV entry and pathogenesis. As a proof-of-concept, we show that the green tea polyphenol epigallocatechin gallate (EGCG) inhibits entry of endemic human CoVs and highly pathogenic emergent and pre-emergent CoVs via inhibition of CoV attachment to cell surface glycans. We observed that the ubiquitous cell surface glycan heparan sulfate appears to be a conserved attachment factor for CoVs, while sialic acid is utilized only for CoVs that require endocytosis for entry. The SARS-CoV-2 spike protein is itself highly glycosylated and these glycosylation sites have remained conserved across variants of concern, highlighting the evolutionary pressure to maintain glycosylation. We determined that complex and high-mannose glycans of SARS-CoV-2 spike are involved in viral entry, which informed the design of multivalent glycan-based dendrimers as CoV entry inhibitors. In parallel, we found an unexpected role for spike glycans in modulating the activation of Toll-like receptor 4 (TLR4) and subsequent proinflammatory host responses, thus implicating spike glycosylation in CoV pathogenesis. We finally expanded our work to another viral glycoprotein, dengue virus non-structural protein 1 (NS1), to further dissect the interplay of viral protein glycosylation, TLR4, and proinflammatory responses. While mutation of NS1 glycosylation sites in dengue virus reduced proinflammatory responses in infected THP-1-derived macrophages in a TLR4-independent manner, we identified a striking role for TLR4 in restricting dengue virus infection. Understanding the conserved roles of glycan-dependent interactions in entry and pathogenesis will inform the development of broad-spectrum antiviral molecules to prepare for future emerging viruses.