Abstract
Coronaviruses (CoVs) are notorious for crossing host species barriers. Their emergence upon zoonotic introduction poses a significant threat to public health as poignantly illustrated by the ongoing SARS-CoV-2 pandemic. Thus, there is an evident need to understand the conditions that facilitate or hamper coronaviruses species jumping. Of four established human
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CoVs, HCoV-OC43 and HCoV-HKU1 use 9-O-Ac-Sia for attachment and both emerged from the same minor clade of embecoviruses. They also differ from all other coronaviruses in that they possess two types of envelope proteins: the receptor binding fusion protein S, and the hemagglutinin-esterase protein (HE) which has receptor-destroying activity. In chapter 2, we provide evidence that, in contrast to the HEs of animal corona- and toroviruses, the HEs of OC43 and HKU1 gradually lost their lectin function during circulation in the human population. We show that the HE lectin domain is a regulator of HE esterase activity and enhances the destruction of clustered glycotopes. Loss of HE lectin function, apparently in combination with the size difference between S and HE, altered virion-associated receptor-destroying activity: it was strongly reduced, became fully dependent on receptor-binding by S and became selective for certain receptor populations. In chapter 3, we took a comparative structural analysis approach in combination with automated ligand docking and mutational analysis to identify the RBS in the β1CoV S protein. We show that this site is not only present in the S proteins of BCoV, HCoV-OC43 and PHEV, but also conserved and functional in HCoV-HKU1. We provide direct evidence that the RBS is essential for virus infectivity by vesicular stomatitis virus pseudotyping. In chapter 4, we extended our findings by engaging in a collaborative study to structurally identify the RBS of HCoV-OC43. A holostructure was determined by cryo-electron microscopy of OC43 S in complex with 9-O-acetylated sialic acid at 2.8 Å resolution. Moreover, we were first to analyze the binding kinetics of the S RBS to 9-O-Ac-Sia in monovalent 1:1 binding assays using biolayer interferometry and synthetic sialosides as ligand. We showed that binding is highly dynamic with association and release occurring within tenths of seconds. In chapter 5, we developed a reverse genetics system for BCoV and, replaying OC43 evolution, used the system to abolish the HE RBS. We show that loss of HE lectin function consistently selects for second-site mutations in the S RBS that dramatically reduce S binding affinity. In some mutants, bivalent binding of S1A-Fc fusion proteins to the clustered sialoglycans of bovine submaxillary mucin was reduced by four orders of magnitude. We present data to demonstrate that HE and S are in functional balance and co-evolve, with selection not only for optimal balance between attachment and receptor-destruction, but also for maximal virion avidity within the given constraints. The data lead us to conclude that the acquisition of an HE gene has set the embecoviruses on an evolutionary path that selected for mechanisms of virus-glycan interactions that are unique among coronaviruses, but remarkably similar to those seen in influenza A viruses.
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