Biology subjects

de Groot, R. J.

Publications and source records attributed to de Groot, R. J..

5 recordsLinked to original sources

bioRxiv2026

Spike and host glycan determinants of HKU1 airway tropism

Human coronavirus HKU1 comprises two distinct serotypes, A and B, whose spike proteins are substantially divergent. Here, we show that spikes from both serotypes preferentially bind 9-O-acetylated 2,8-linked disialosides. Cryo-electron microscopy of the B-type N5 spike reveals a conserved extended binding site in domain S1A that accommodates both the terminal and penultimate sialic acid residues, with interactions involving the penultimate residue substantially enhancing binding. Spike N-glycan processing modulates affinity and linkage selectivity; glycans flanking the binding pocket offer a plausible structural basis for these effects. In contrast to the HKU1-A spike, which adopts open S1B-up conformations upon ligand binding, the N5 apo structure showed that the ligand-binding site was already formed and the e1 relay element register-shifted in most protomers. Nevertheless, we detected neither spontaneous opening nor a transition to an S1B-up state following ligand binding. These findings, obtained with a minimally modified ectodomain, differ from recent reports of ligand-independent opening. Molecular dynamics simulations indicated that membrane-embedded GT3, but not GD3, presents its glycan chain in a geometry compatible with S1A-mediated engagement. Concordantly, in human nasal epithelial cultures cell surface GT3-like O-acetylated trisialoside glycotopes were detected in ciliated cells, linking their cell-type-specific presentation to HKU1 tropism.

microbiology↗

bioRxiv2023

Sialoglycan binding triggers spike opening in a human coronavirus

Coronavirus (CoV) spikes mediate receptor binding and membrane fusion, making them prime targets for neutralising antibodies. In the cases of SARS-CoV, SARS-CoV-2, and MERS-CoV, spikes transition freely between open and closed conformations to balance host cell attachment and immune evasion. The open conformation exposes domain S1B, allowing it to bind to proteinaceous cell surface receptors. It also facilitates protein refolding during spike-mediated membrane fusion. However, with a single exception, the pre-fusion spikes of all other CoVs studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, where spikes more commonly transition to open states in response to specific cues, rather than spontaneously. In our study, using cryo-EM and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes upon binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state via allosteric inter-domain cross-talk. Our findings paint a more elaborate picture of CoV attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

microbiology↗

bioRxiv2022

SARS-CoV-2 nucleocapsid protein inhibits the stress response through RNA-binding domain N2b

The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding - prevented PKR activation, inhibited {beta}-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera. SIGNIFICANCE STATEMENTSARS-CoV-2 nucleocapsid protein N is an antagonist of innate immunity but how it averts virus detection by intracellular sensors remains subject to debate. We provide evidence that SARS-CoV-2 N, by sequestering dsRNA through domain N2b, prevents PKR-mediated activation of the integrated stress response as well as detection by RIG-I-like receptors and ensuing type I interferon expression. This function, conserved in human coronavirus 229E, is not affected by mutations that prevent posttranslational modifications, previously implicated in immune evasion, or that target its binding to stress granule scaffold proteins. Our findings further our understanding of how SARS-CoV-2 evades innate immunity, how this may drive viral evolution and why increased N expression may have been a selective advantage to SARS-CoV-2 variants of concern.

microbiology↗

bioRxiv2020

Coronavirus hemagglutinin-esterase and spike proteins co-evolve for functional balance and optimal virion avidity

Human coronaviruses OC43 and HKU1 are respiratory pathogen of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spill-over. 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 towards 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 selected for 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 co-evolve 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 (IAVs). Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

microbiology↗

bioRxiv2020

Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans

The human betacoronaviruses HKU1 and OC43 (subgenus Embecovirus) arose from separate zoonotic introductions, OC43 relatively recently and HKU1 apparently much longer ago. Embecovirus particles are studded with two types of surface projections called S (for spike) and HE (for haemagglutinin-esterase), with S mediating receptor-binding and membrane fusion and HE acting as a receptor-destroying enzyme. Together, they promote dynamic virion attachment to glycan-based receptors with 9-O-acetylated sialic acid as main constituent. We recently showed that adaptation of HKU1 and OC43 to replication in the human respiratory tract involved loss-of-function mutations in the lectin domain of HE. Here we present the cryo-EM structure of the [~]80 kDa, heavily glycosylated HKU1 HE at a global resolution of 3.4 [A]. Comparison to existing HE structures reveals a drastically truncated lectin domain, incompatible with sialic acid binding, but with the structure and function of the HE esterase domain left intact. Our cryo-EM structure, in combination with mass spectrometry analysis, also describes the extent of glycosylation on the now redundant lectin domain, which forms a putative glycan shield. The findings further our insight into the evolution and host adaptation of human embecoviruses and also demonstrate the utility of cryo-EM for studying small, heavily glycosylated proteins which are intractable to X-ray crystallography.

microbiology↗