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The Murine Coronavirus Hemagglutinin-esterase Receptor-binding Site: A Major Shift in Ligand Specificity through Modest Changes in Architecture

  • Martijn A. Langereis ,

    Contributed equally to this work with: Martijn A. Langereis, Qinghong Zeng

    Affiliation Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

  • Qinghong Zeng ,

    Contributed equally to this work with: Martijn A. Langereis, Qinghong Zeng

    Affiliation Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Sciences, Utrecht University, Utrecht, The Netherlands

  • Balthasar Heesters,

    Affiliation Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

  • Eric G. Huizinga ,

    e.g.huizinga@uu.nl (EGH); r.j.degroot@uu.nl (RJdG)

    Affiliation Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Sciences, Utrecht University, Utrecht, The Netherlands

  • Raoul J. de Groot

    e.g.huizinga@uu.nl (EGH); r.j.degroot@uu.nl (RJdG)

    Affiliation Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

The Murine Coronavirus Hemagglutinin-esterase Receptor-binding Site: A Major Shift in Ligand Specificity through Modest Changes in Architecture

  • Martijn A. Langereis, 
  • Qinghong Zeng, 
  • Balthasar Heesters, 
  • Eric G. Huizinga, 
  • Raoul J. de Groot
PLOS
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Correction

22 Feb 2012: Langereis MA, Zeng Q, Heesters BA, Huizinga EG, de Groot RJ (2012) Correction: The Murine Coronavirus Hemagglutinin-esterase Receptor-binding Site: A Major Shift in Ligand Specificity through Modest Changes in Architecture. PLOS Pathogens 8(2): 10.1371/annotation/a1e2a2e4-df03-40db-b10b-fd0cfcf78d3c. https://doi.org/10.1371/annotation/a1e2a2e4-df03-40db-b10b-fd0cfcf78d3c View correction

Abstract

The hemagglutinin-esterases (HEs), envelope glycoproteins of corona-, toro- and orthomyxoviruses, mediate reversible virion attachment to O-acetylated sialic acids (O-Ac-Sias). They do so through concerted action of distinct receptor-binding (“lectin”) and receptor-destroying sialate O-acetylesterase (”esterase”) domains. Most HEs target 9-O-acetylated 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-acetylated Sias instead. Here we present the crystal structure of the lectin domain of mouse hepatitis virus (MHV) strain S HE, resolved both in its native state and in complex with a receptor analogue. The data show that the shift from 9-O- to 4-O-Ac-Sia receptor usage primarily entailed a change in ligand binding topology and, surprisingly, only modest changes in receptor-binding site architecture. Our findings illustrate the ease with which viruses can change receptor-binding specificity with potential consequences for host-, organ and/or cell tropism, and for pathogenesis.

Author Summary

Glycans cover the surface of every living cell. In vertebrates, these sugar trees commonly terminate with sialic acid (Sia) and, in consequence, Sias have become the attachment factors of choice for a multitude of pathogens: protozoa, bacteria and viruses alike. To ensure selectivity, viruses evolved to target distinct Sia species. Whether a particular type of Sia serves as receptor may depend -amongst others- on the absence or presence of specific Sia modifications. For example, most group A betacoronaviruses attach to 9-O-acetylated Sias. However, some murine coronaviruses have switched to using 4-O-acetylated Sias instead. In chemical/molecular terms this represents a momentous shift in receptor usage. We now have crystallized the hemagglutinin-esterase protein (HE) of a murine coronavirus and have solved the structure of its sugar-binding domain. Our findings reveal in exquisite detail the interactions between Sia binding site and cognate receptor. The data allow a reconstruction of how, during coronavirus evolution, the switch in receptor usage may have come about.

Introduction

To initiate infection viruses must bind to an appropriate host cell. Selectivity of binding is ensured by attachment proteins on the virion, tailored to recognize one -or at the most- a limited number of cell surface molecules. Remarkably, a large number of viruses, representative of at least 11 distinct families several of which of clinical and/or veterinary importance, use sialic acid (Sia) as receptor determinant. Owing to differential modification, Sia structural diversity exceeds that of any other monosaccharide [1]. The most common type of Sia substitution, O-acetylation at carbon atoms C4, C7, C8 and/or C9, occurs in a host-, organ- and even cell-specific fashion such that even individual cells of the same type and tissue may differ in their Sia expression profile [2][4]. Viruses have evolved to selectively use particular Sia variants and their attachment proteins are high-specificity sialolectins, the binding of which might depend on the identity of the penultimate residue in the sugar chain, the type of glycosidic linkage and/or the presence or absence of substitutions [5][9]. Ultimately, this preference in Sia receptor usage affects host-, organ-, and cell-tropism [10][14], the course and outcome of infection [15][18] as well as the efficacy of intra- and cross-species transmission [14], [19], all to extents not yet fully appreciated.

The hemagglutinin-esterases (HEs) are a class of Sia-binding envelope glycoproteins found in some negative-stranded RNA viruses, namely in influenza C and infectious salmon anemia virus (family Orthomyxoviridae; [5], [20]), but also in toro- and coronaviruses, positive-stranded RNA viruses in the order Nidovirales [21], [22]. From phylogenetic and comparative structural analyses it appears that toro- and coronaviruses acquired their HE proteins separately via horizontal gene transfer, with an (hemagglutinin-esterase-fusion) HEF-like protein as progenitor [22][25]. Like influenza C virus HEF, most nidovirus HEs bind to 9-O-acetylated (9-O-Ac) Sias and, correspondingly, display sialate-9-O-acetylesterase receptor-destroying enzyme activity [25]. Murine coronaviruses, however, occur in two closely related biotypes that differ in HE ligand/substrate preference. One of these -represented by mouse hepatitis virus (MHV) strain DVIM- displays the presumptive ancestral specificity and targets 9-O-Ac-Sias, while the other -represented by MHV strain S- appears to have evolved to use 4-O-Ac-Sias instead [6], [25][27] (for supplementary introduction see Text S1 and Figure S1). Given the stereochemical differences between these Sia variants (Figure 1) and the essentially different requirements for ligand and substrate recognition by the respective HEs, the question arises how this major shift in receptor usage was achieved and what changes must have occurred in the receptor-binding and O-acetylesterase domains to make this transition possible.

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Figure 1. Stereochemical differences between 9-O- and 4-O-acetylated sialic acid.

Stick representation of (left) αNeu5,9Ac22Me and (right) αNeu4,5Ac22Me. Backbone αNeu5Ac2Me is colored in gray (carbon), red (oxygen) and blue (nitrogen). The 9-O-Ac group of αNeu5,9Ac22Me and 4-O-Ac group of αNeu4,5Ac22Me are highlighted in cyan (carbon).

https://doi.org/10.1371/journal.ppat.1002492.g001

The crystal structures of a number of 9-O-Ac-Sia-specific nidovirus HEs have been solved [23], [24]. Unlike the receptor-binding site (RBS) of influenza C virus HEF [28], the RBSs of the corona- and torovirus HEs seem to be exceptionally plastic as they appear to have undergone significant changes and adaptations that altered their overall architecture in a relatively short evolutionary time span. Based on these observations, we anticipated and speculated [23] that this plasticity might have allowed for even more substantial adjustments in the RBS of the murine coronavirus HE as to produce an entirely novel binding site specific for 4-O-acetylated Sias.

We now present the crystal structure of the MHV-S HE receptor-binding domain, both in its native state and in complex with a receptor analogue. The data reveal in exquisite detail how the RBS changed to accommodate 4-O- instead of 9-O-acetylated Sias. Surprisingly, however, this shift in receptor usage seems to have involved primarily a change in ligand binding topology and relatively modest changes in RBS architecture.

Results/Discussion

Expression, purification, and biochemical characterization of MHV-S HE

We produced the ectodomain of MHV-S HE (residues 25-403) as an Fc-fusion protein, either in enzymatically active (HE-Fc) or inactive form (HE0-Fc), by transient transfection of HEK293 cells. MHV-S HE0-Fc bound to horse serum glycoproteins (HSG), which are decorated with 4-O-acetylated sialic acids (4-O-Ac-Sia), but carry little to no 9-O-Ac-Sias (Figure 2A; [29]). The receptor determinants in HSG could be destroyed by treatment with MHV-S HE-Fc, but not by treatment with BCoV-Mebus HE-Fc (a sialate-9-O-acetylesterase; Figure 2B). No binding of MHV-S HE0-Fc was observed to bovine submaxillary mucin (BSM), a glycoconjugate devoid of 4-O-Ac-Sias (Figure 2A; [30]). The MHV-S HE ectodomain, released from HE-Fc by thrombin-cleavage, retained proper sialate-4-O-acetylesterase activity when assayed for substrate specificity with a synthetic di-O-acetylated Sia (5-N-acetyl-4,9-di-O-acetylneuraminic acid α-methylglycoside, αNeu4,5,9Ac32Me; Figure 2C). In hemagglutination assays, MHV-S HE0 specifically bound to 4-O-acetylated Sias (Figure 2D). The combined findings show that the recombinant MHV-S HE proteins are biologically active, both as Fc fusion proteins (Figures 2A and B) and after the removal of the Fc tail by thrombin-cleavage (Figures 2C and D), which we take as an indication for proper folding and protein stability.