Biology subjects

Hulswit, R. J. G.

Publications and source records attributed to Hulswit, R. J. G..

6 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↗

bioRxiv2025

Cetacean coronavirus spikes highlight S glycoprotein structural plasticity

Coronaviruses (CoVs) exhibit a remarkable ability for spill-over infections into naive host populations. While much research has focused on the spike (S) glycoproteins of zoonotic alpha- and betacoronaviruses, the S proteins of gamma- and deltacoronaviruses, which predominantly infect avian hosts, remain poorly understood. Here, we present high-resolution cryo-EM structures of S proteins from two distinct gammacoronaviruses (75.7% sequence identity) that atypically infect marine mammals and belong to the Gammacoronavirus delphinapteri species. The cryo-EM reconstructions reveal that the spikes exhibit a unique quaternary architecture that distinguishes them from other coronaviruses. The S protein features a previously unidentified, tripodal quaternary assembly of the S1 subunit, in which S1B domains are presented in an upright position while their putative receptor binding sites are shielded by extended loops from the S1A domain of the same protomers. Additionally, the CeCoV spike proteins have evolved an additional and unique [~]200 residue N-terminal domain (S10). S10 lacks homology to known protein sequences but displays structural similarity to members of the cupin protein superfamily. This represents a remarkable case of coronaviral exaptation of a host protein integrated into the S glycoprotein. Moreover, glycoproteomic analyses reveal that CeCoV S proteins are extensively N-glycosylated (>100 N-glycans per trimer), with a notable abundance of high-mannose glycans on S10 and O-glycosylation sites within a mucin-like loop at the trimer apex, all contributing to a dense glycan shield and potentially masking immunogenic epitopes. These findings demonstrate the structural diversity and adaptability of CoV S proteins, including alternative quaternary assemblies, additional domains, and diverse glycosylation strategies, offering new insights into the evolutionary mechanisms that enable coronaviruses to expand their host range and establish infections in novel species.

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↗

bioRxiv2021

Identification of site-specific evolutionary trajectories shared across human betacoronaviruses

Comparing the evolution of distantly related viruses can provide insights into common adaptive processes related to shared ecological niches. Phylogenetic approaches, coupled with other molecular evolution tools, can help identify mutations informative on adaptation, whilst the structural contextualization of these to functional sites of proteins may help gain insight into their biological properties. Two zoonotic betacoronaviruses capable of sustained human-to-human transmission have caused pandemics in recent times (SARS-CoV-1 and SARS-CoV-2), whilst a third virus (MERS-CoV) is responsible for sporadic outbreaks linked to animal infections. Moreover, two other betacoronaviruses have circulated endemically in humans for decades (HKU1 and OC43). To search for evidence of adaptive convergence between established and emerging betacoronaviruses capable of sustained human-to-human transmission (HKU1, OC43, SARS-CoV-1 and SARS-CoV-2), we developed a methodological pipeline to classify shared non-synonymous mutations as putatively denoting homoplasy (repeated mutations that do not share direct common ancestry) or stepwise evolution (sequential mutations leading towards a novel genotype). In parallel, we look for evidence of positive selection, and draw upon protein structure data to identify potential biological implications. We find 30 mutations, with four of these [codon sites 18121 (nsp14/residue 28), 21623 (spike/21), 21635 (spike/25) and 23948 (spike/796); SARS-CoV-2 genome numbering] displaying evolution under positive selection and proximity to functional protein regions. Our findings shed light on potential mechanisms underlying betacoronavirus adaptation to the human host and pinpoint common mutational pathways that may occur during establishment of human endemicity.

evolutionary biology↗

bioRxiv2020

Molecular rationale for hantavirus neutralization by a reservoir host-derived monoclonal antibody

The intricate lattice of Gn and Gc glycoprotein spike complexes at the surface of hantaviruses facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Here, through study of a neutralizing monoclonal antibody (mAb 4G2) generated in a bank vole reservoir host following infection with Puumala virus (PUUV), we provide molecular-level insights into how antibody-mediated targeting of the hantaviral glycoprotein lattice effectively neutralizes the virus. Crystallographic analysis reveals that mAb 4G2 binds to a multi-domain site on Gc in the pre-fusion state, and that Fab binding is incompatible with the conformational changes of the Gc that are required for host cell entry. Cryo-electron microscopy of PUUV-like particles treated with Fab 4G2 demonstrates that the antibody binds to monomeric Gc at breaks in the Gn-Gc lattice, highlighting the immunological accessibility of Gc monomers on the mature hantavirus surface and the plastic nature of the higher-order lattice assembly. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.

microbiology↗

bioRxiv2020

Identification of a common deletion in the spike protein of SARS-CoV-2

Two notable features have been identified in the SARS-CoV-2 genome: (1) the receptor binding domain of SARS-CoV-2; (2) a unique insertion of twelve nucleotide or four amino acids (PRRA) at the S1 and S2 boundary. For the first feature, the similar RBD identified in SARs-like virus from pangolin suggests the RBD in SARS-CoV-2 may already exist in animal host(s) before it transmitted into human. The left puzzle is the history and function of the insertion at S1/S2 boundary, which is uniquely identified in SARS-CoV-2. In this study, we identified two variants from the first Guangdong SARS-CoV-2 cell strain, with deletion mutations on polybasic cleavage site (PRRAR) and its flank sites. More extensive screening indicates the deletion at the flank sites of PRRAR could be detected in 3 of 68 clinical samples and half of 22 in vitro isolated viral strains. These data indicate (1) the deletion of QTQTN, at the flank of polybasic cleavage site, is likely benefit the SARS-CoV-2 replication or infection in vitro but under strong purification selection in vivo since it is rarely identified in clinical samples; (2) there could be a very efficient mechanism for deleting this region from viral genome as the variants losing 23585-23599 is commonly detected after two rounds of cell passage. The mechanistic explanation for this in vitro adaptation and in vivo purification processes (or reverse) that led to such genomic changes in SARS-CoV-2 requires further work. Nonetheless, this study has provided valuable clues to aid further investigation of spike protein function and virus evolution. The deletion mutation identified in vitro isolation should be also noted for current vaccine development.

genetics↗