In 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) was discovered as the causative agent of a respiratory disease with an unusually high fatality rate of ~35%. For comparison, the yearly seasonal flu has a fatality rate of ~0.1%. This infection is still ongoing with no vaccine or anti-viral therapeutics available. There are five other known human-infecting CoVs, including the infamous severe acute respiratory syndrome coronavirus (SARS-CoV) and four endemic viruses mostly responsible for mild respiratory infections with common cold-like symptoms. MERS-CoV is a zoonotic virus that can infect both animals, like camels or bats, and humans. MERS-CoV is an enveloped virus with protrusions or spikes extending from its surface to promote both viral attachment and initiation of infection through fusion of the viral and host cell membranes (Figure 1). Two of the many open questions about coronaviruses that we set out to answer in our article was: how coronaviruses attach to their host cells and how the viral host specificity, or tropism, could change.
MERS-CoV infects human lung epithelial cells via interactions with cell surface sialosides and dipeptidyl-peptidase 4 that serve as attachment and entry receptors, respectively (1). Although sialic acid derivatives are ubiquitous on host-cells, they exhibit a wide diversity depending on chemical modifications (e.g. acetylation) or glycosidic linkages (e.g. a-2,3 versus a-2,6 bonds) that can participate in both the specificity of interactions with pathogens and selection of target cells. Influenza viruses also use sialosides as their major receptor. We previously used cryo-electron microscopy (cryoEM) to visualize a sialoside bound to the spike glycoprotein of an endemic CoV that causes mild respiratory infection (2). Here, we set out to first identify how the MERS-CoV spike binds sialic acid using cryoEM. We tackled this problem using cryoEM grids covered with a home-made thin layer of carbon to prevent preferred specimen orientation upon vitrification which typically plagues MERS-CoV spike structural studies. The outcome was a beautiful 2.7Å resolution structure revealing the ~300 dalton sialoside and the interactions it makes with the MERS-CoV spike glycoprotein (Figure 2) that are distinct from those observed previously for OC43 (2). These results distinguish CoV spikes from influenza virus hemagglutinins as the latter glycoproteins all bind sialic acids in a similar manner.
To assess the importance of the identified interactions for viral entry into human epithelial cells, we tested MERS-CoV pseudovirions (non-replicative viruses) ability to enter cells. Pseudoviruses with single point mutations in the binding site could no longer enter cells, showing that sialic acid binding via the identified site is a necessary step for viral entry.
We next used cryoEM to illuminate the molecular determinants of the MERS-CoV binding selectivity for sialosides and understand their contribution to host specificity or tropism. A hint that sialoside specificity plays a role in tropism: MERS-CoV is unable to infect horses even though the equine receptor allows viral entry into host cells in vitro (4,5,6) This suggested a putative role of sialosides in modulating the range of target hosts. Our structures of MERS-CoV spike in complex with sialyl-oligosaccharides suggest that preferred interactions with a2,3-linked over a2,6-linked sugars result from enhanced contacts with the former class of ligands and that glycolyl containing sialosides are sterically disfavored from binding to the pocket. Horse respiratory tracts are decorated with glycolyl rich sialosides (7), giving a plausible explanation for the lack of MERS-CoV infection in horses.
This study identified the location and interactions of the MERS-CoV spike with sialoside receptors, demonstrated these interactions are required for MERS-CoV spike-mediated entry into human airway epithelial cells, and shed light on the molecular intricacies of the changing host range and tropism of this virus based on sialoside interactions.
1. Raj VS et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature. 495, 251-254 (2013). 2. Tortorici MA et al. Structural basis for human coronavirus attachment to sialic acid receptors. Nat Struct Mol Biol. 26, 481-489 (2019). 3. Goddard TD et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14-25 (2018). 4. Barlan, A et al. Receptor variation and susceptibility to Middle East respiratory syndrome coronavirus infection. J. Virol. 88, 4953–4961 (2014). 5. Meyer, B et al. Serologic assessment of possibility for MERS-CoV infection in equids. Emerg. Infect. Dis. 21, 181–182 (2015). 6. Vergara-Alert, J et al. Livestock susceptibility to infection with Middle East respiratory syndrome coronavirus. Emerg. Infect. Dis. 23, 232–240 (2017). 7. Suzuki, Y. et al. Sialic acid species as a determinant of the host range of influenza A viruses. J. Virol. 74, 11825–11831 (2000).