Viral hemagglutinin-esterases: Mediators of dynamic virion-glycan interactions

Abstract

The sialic acids (Sias), a diverse family of 9-carbon sugars, are among the most important molecules of life. Commonly occurring as terminal residues of glycans on proteins and lipids, they are key elements of glycotopes of cellular lectins and there is accumulating evidence for them to act as chemical messengers in cell recognition and signaling events. Their function in such interactions may be modulated through specific modifications, the most common of which is O-acetylation at Sia carbon atoms C4, C7, C8 and/or C9. These modified Sias have not been studied in great detail, at least in part because of technical hurdles and limitations associated with the analysis of these elusive sugars. Viruses that use Sias as receptor determinant come with a treasure trove of lectins and Sia-modifying enzymes. Hemagglutinin-esterases (HEs), closely related envelope glycoproteins in influenza C, corona- and toroviruses, mediate reversible attachment to O-acetylated (O-Ac) Sias. They do so through concerted action of distinct receptor-binding and receptor-destroying sialate O-acetylesterase domains. Most HEs target 9-O-Ac-Sias. In one lineage of murine coronaviruses, however, HE esterase substrate and lectin ligand specificity changed dramatically as these viruses evolved to use 4-O-Ac-Sias instead. Torovirus HEs also differ in receptor usage, although more subtle. While porcine torovirus HEs recognize 9-O-Ac-Sias, bovine torovirus HEs have a preference for 7,9-di-O-Ac-Sias. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. In this thesis we present crystal structures of bovine coronavirus HE, porcine and bovine torovirus HEs and the receptor-binding site of a murine coronavirus HE in complex with receptor-analogues. We show that both corona- and torovirus HEs arose from an influenza C-like hemagglutinin-esterase fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, while remnants of the fusion domain were adapted to establish novel monomer-monomer contacts. Structure-guided biochemical analysis of the torovirus esterase domains revealed that a functionally, but not structurally conserved Arginine-Sia carboxylate interaction is critical for the binding and positioning of glycosidically-bound Sias in the catalytic pocket. The distinct preference of the porcine torovirus enzyme for 9-mono- over 7,9-di-O-acetylated Sias can be explained from a single residue difference. The structures of the receptor binding domains of bovine coronavirus and torovirus HEs are dramatically different although they bind all their ligand in a 9-O-Ac dependent way. The major shift from 9-O- to 4-O-Ac-Sia receptor usage in murine coronaviruses primarily entailed a change in ligand binding topology and, surprisingly, only modest changes in receptor-binding site architecture. Biochemical analysis of a broad spectrum of corona- and torovirus HEs revealed that porcine torovirus strain P4, bovine coronavirus strain LUN and mouse hepatitis virus strain S bound 9-mono-O-, 7,9-di-O-, and 4-O-Ac-Sias, respectively, with a very high binding affinity and specificity. These virolectins allowed specific detection of and distinction between closely related O-Ac-Sias in mammalian cells and tissues. Differential expression of these Sias suggests that regulation of Sia-O-acetylation is crucial to the development, homeostasis and function of many organs and tissues.