Coronaviruses cause respiratory tract infections in humans and outbreaks of deadly pneumonia worldwide. Infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host receptors and fuses the viral and cellular membranes. To understand the molecular basis of coronavirus attachment to oligosaccharide receptors, we determined cryo-EM structures of coronavirus OC43 S glycoprotein trimer in isolation and in complex with a 9-O-acetylated sialic acid. We show that the ligand binds with fast kinetics to a surface-exposed groove and that interactions at the identified site are essential for S-mediated viral entry into host cells, but free monosaccharide does not trigger fusogenic conformational changes. The receptor-interacting site is conserved in all coronavirus S glycoproteins that engage 9-O-acetyl-sialogycans, with an architecture similar to those of the ligand-binding pockets of coronavirus hemagglutinin esterases and influenza virus C/D hemagglutinin-esterase fusion glycoproteins. Our results demonstrate these viruses evolved similar strategies to engage sialoglycans at the surface of target cells.

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion–sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

Extracellular vesicles (EV) that are released by immune cells are studied intensively for their functions in immune regulation and are scrutinized for their potential in human immunotherapy, for example against cancer. In our search for signals that stimulate the release of functional EV by dendritic cells we observed that LPS-activated human monocyte-derived dendritic cells (moDC) changed their morphological characteristics upon contact with non-cognate activated bystander T-cells, while non-activated bystander T-cells had no effect. Exposure to activated bystander T-cells also stimulated the release of EV-associated proteins by moDC, particularly CD63, and ICAM-1, although the extent of stimulation varied between individual donors. Stimulation of moDC with activated bystander T-cells also increased the release of EV-associated miR155, which is a known central modulator of T-cell responses. Functionally, we observed that EV from moDC that were licensed by activated bystander T-cells exhibited a capacity for antigen-specific T-cell activation. Taken together, these results suggest that non-cognatei interactions between DC and bystander T-cells modulates third party antigen-specific T-cell responses via EV.

22Human coronaviruses OC43 and HKU1 are respiratory pathogen of zoonotic origin that have gained 23 worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spill-over. All 24 three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase HE 25 acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity 26 towards clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to 27 humans. Replaying OC43 evolution, we knocked-out BCoV HE lectin function and performed forced 28 evolution-population d ynamics analysis. Loss of HE receptor-binding selected for second-site 29 mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations 30 selected for cooperativity in virus swarms with low-affinity S minority variants sustaining propagation 31 of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site 32 substitutions in both S and HE. Apparently, S and HE are functionally interdependent and co-evolve to 33 optimize the balance between attachment and release. This mechanism of glycan-based receptor 34 usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among 35 CoVs, but reminiscent of that of influenza A viruses (IAVs). Apparently, general principles fundamental 36 to virion-sialoglycan interactions prompted convergent evolution of two important groups of human 37 and animal pathogens. 38(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 39The subfamily Orthocoronavirinae comprises a group of enveloped positive-strand RNA viruses of 40 clinical and veterinary significance. Adding to the socio-economic impact of coronaviruses (CoVs) 41 already extant in humans and livestock, the emergence of 'new' CoVs through cross species 42 transmission poses an ever-looming threat to public health, animal health, and food production. 43 Seven coronaviruses are known to infect humans, but not all of them have become established. The 44 introduction of SARS CoV in 2002 from horseshoe bats with masked palm civets as incidental transient 45 hosts, was rapidly contained through quarantine measures 1 . MERS CoV, natural to dromedary camels, 46 causes a classical zoonotic infection with limited human-to-human spread 2 . December 2019, a 47 member of the species Severe acute respiratory syndrome related coronavirus (SARS-CoV), called 48 SARS-CoV-2 and 79.5% identical to the 2002 SARS CoV variant, emerged in Wuhan, China 3,4 to progress 49 to full scale pandemicity. Chances are, SARS-CoV-2 will eventually become established in the human 50 population. 51Four other respiratory coronaviruses of zoonotic origin already did succeed in becoming true human 52 viruses with world-wide distribution 5-7 . Among them are HKU1 and OC43 (subgenus Embecovirus, 53 genus Betacoronavirus), related yet distinct viruses that arose from diff...

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