Abstract
Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.
Highlights
SARS-CoV-2 has sparked a society changing pandemic, and additional means to understand this virus will facilitate counter-measures
Receptor binding of SARS-CoV-2 fluorescent trimeric receptor-binding domain (RBD) proteins imaging work in the Ward lab was supported by Bill and Melinda Gates Foundation OPP1170236
The results demonstrate that trimeric fully glycosylated RBD trimers are excellent tools to study receptor binding properties
Summary
SARS-CoV-2 has sparked a society changing pandemic, and additional means to understand this virus will facilitate counter-measures. Several proteinases induce the spike protein into its fusogenic form allowing the fusion of the viral and target membrane. This pathway is known, the details that are of importance for receptor binding and what differentiates SARS-CoV-2 from SARS-CoV-1, are incompletely understood. It has been shown that the affinity of the SARS-CoV-2 spike to ACE2 is significantly higher compared to SARS-CoV-1 [2,4]. How this relates to tissue and cell tropism remains to be determined
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