Biology subjects

Hurdiss, D. L.

Publications and source records attributed to Hurdiss, D. L..

17 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↗

bioRxiv2026

Structure of SARS-CoV-2 spike in complex with its co-receptor the neuronal cell adhesion protein contactin 1

The emergence of SARS-CoV-2 has caused millions of deaths and excess morbidity in the worldwide population. In addition to its respiratory symptoms, SARS-CoV-2 has become known for its neurotropism and long-term neurological sequelae, with a post-acute infection syndrome commonly referred to as long-COVID. Next to the host receptor angiotensin-converting enzyme 2 (ACE2) additional interactions of the SARS-CoV-2 spike (S) protein have been described for neuronal co-receptors specific to the nervous system including cell adhesion protein contactin 1 (CNTN1). Details of the spike-CNTN1 interaction have remained elusive. Here, we quantified the spike-CNTN1 interaction by surface plasmon resonance and resolved the structure of the complex by single particle cryo-electron microscopy (cryo-EM). Spike and CNTN1 interact with nanomolar affinity, driven by an avidity effect and mediated by the horseshoe moiety of CNTN1. The cryo-EM structure reveals that the CNTN1 Ig1-4 horseshoe is wedged in between two receptor binding domains (RBDs) and interacts, through Ig3, with a unique receptor interface at the base of the RBD in the up-conformation. This receptor interface is not previously described for other spike receptors but overlaps with the epitopes of several neutralizing monoclonal antibodies. Comparison of our data with available spike structures suggests one spike trimer can bind three CNTN1 molecules, or alternatively, different co-receptors such as ACE2 and CNTN1, simultaneously. These findings shed new light on the molecular determinants of SARS-CoV-2 neurotropism.

biochemistry↗

bioRxiv2025

The gH/gL/gO Complex of Elephant Endotheliotropic Herpesvirus 1A Functions as a Receptor Binding Complex

Elephant endotheliotropic herpesvirus (EEHV) causes a fatal haemorrhagic disease in young elephants and represents a major threat to the survival of these endangered species. Despite its clinical importance, the mechanisms of EEHV entry and host cell recognition remain poorly understood. Here, we show that the EEHV1A homolog of the Betaherpesvirus glycoprotein O (gO) assembles with glycoproteins H and L (gH/gL) to form a heterotrimeric complex analogous to the human cytomegalovirus (HCMV) gH/gL/gO trimer. Using single-particle cryo-electron microscopy, we determined the structure of the EEHV1A gH/gL/gO complex at 3.2 [A] resolution, revealing a conserved overall architecture but distinct gO topology and disulfide arrangement. The EEHV1A trimer, but not gH/gL alone, bound primary elephant endothelial cells, monocytes, and lymphocytes, indicating that gH/gL/gO serves as the receptor-binding complex of EEHV1A. This binding was specifically blocked by sera from naturally infected elephants, confirming its antigenic relevance. Finally, negative-stain electron microscopy polyclonal epitope mapping of gH/gL/gO-Fab complexes identified a Fab population that bound gO near the apex of the gH/gL/gO trimer, suggesting that these antibodies may interfere with gH/gL/gO-receptor interactions. Together, these findings define the molecular architecture and receptor-binding properties of the EEHV1A gH/gL/gO complex and provide a structural framework for the development of vaccines and antibody-based interventions against EEHV-associated disease.

microbiology↗

bioRxiv2025

Integrative Structure of Norovirus NS3 Suggests a Role in RNA Transport

Human noroviruses (HuNoVs) are the leading global cause of non-bacterial gastroenteritis, yet no vaccines or antiviral therapies are currently approved1. The viral non-structural protein NS3 is a membrane-bound AAA+ ATPase of superfamily 3 (SF3) with multiple proposed roles in the norovirus replication cycle. However, the structure of NS3, and the mechanisms by which it contributes to genome replication and membrane remodeling, have remained unknown. We engineered a soluble, hexameric, and catalytically active form of NS3 and determined its cryo-EM structure in the presence of a nucleotide analogue at 2.9 [A] resolution. The structure adopts a split lock-washer architecture characteristic of AAA+ motors that operate via a hand-over-hand translocation mechanism2. Modeling of the nucleotide-binding site reveals conserved features governing ATP binding and hydrolysis. Using integrative modeling with AlphaFold3, we generated a full-length, membrane-associated model of NS3, in which an N-terminal transmembrane domain, central helical bundle, and AAA+ motor form a continuous conduit. This model supports a role for NS3 as a candidate membrane-spanning RNA translocase that couples ATP hydrolysis to genome movement. This structural framework helps address long-standing gaps in our understanding of norovirus replication and establishes a basis for mechanistic studies and structure-guided antiviral design.

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↗

bioRxiv2024

Computational design of developable therapeutic antibodies: efficient traversal of binder landscapes and rescue of escape mutations

Developing therapeutic antibodies is a challenging endeavour, often requiring large-scale screening to produce initial binders, that still often require optimisation for developability. We present a computational pipeline for the discovery and design of therapeutic antibody candidates, which incorporates physics- and AI-based methods for the generation, assessment, and validation of developable candidate antibodies against diverse epitopes, via efficient few-shot experimental screens. We demonstrate that these orthogonal methods can lead to promising designs. We evaluated our approach by experimentally testing a small number of candidates against multiple SARS-CoV-2 variants in three different tasks: (i) traversing sequence landscapes of binders, we identify highly sequence dissimilar antibodies that retain binding to the Wuhan strain, (ii) rescuing binding from escape mutations, we show up to 54% of designs gain binding affinity to a new subvariant and (iii) improving developability characteristics of antibodies while retaining binding properties. These results together demonstrate an end-to-end antibody design pipeline with applicability across a wide range of antibody design tasks. We experimentally characterised binding against different antigen targets, developability profiles, and cryo-EM structures of designed antibodies. Our work demonstrates how combined AI and physics computational methods improve productivity and viability of antibody designs.

bioinformatics↗

bioRxiv2024

A Spike Trimer Dimer-Inducing Nanobody with Anti-Sarbecovirus Activity

The continued emergence and zoonotic threat posed by coronaviruses highlight the urgent need for effective antiviral strategies with broad reactivity to counter new emerging strains. Nanobodies (or single-domain antibodies) are promising alternatives to traditional monoclonal antibodies, due to their small size, cost-effectiveness and ease of bioengineering. Here, we describe 7F, a llama-derived nanobody, targeting the spike receptor binding domain of sarbecoviruses and SARS-like coronaviruses. 7F demonstrates potent neutralization against SARS-CoV-2 and cross-neutralizing activity against SARS-CoV and SARS-like CoV WIV16 pseudoviruses. Structural analysis reveals 7Fs ability to induce the formation of spike trimer dimers by engaging with two SARS-CoV-2 spike RBDs, targeting the highly conserved class IV region. Bivalent 7F constructs substantially enhance neutralization potency and breadth, up to more recent SARS-CoV-2 variants of concern. Furthermore, we demonstrate the therapeutic potential of 7F against SARS-CoV-2 in the fully differentiated 3D tissue cultures mirroring the epithelium of the human airway ex vivo. The broad sarbecovirus activity and distinctive structural features of 7F underscore its potential as promising antiviral against emerging and evolving sarbecoviruses.

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↗

bioRxiv2023

The virus-encoded ion channel "viroporin" activity of the agnoprotein is required for BK Polyomavirus release from infected kidney cells

BK polyomavirus (BKPyV) is a common opportunistic pathogen and the causative agent of several diseases in transplant patients and the immunosuppressed. Despite its importance, aspects of the virus lifecycle such as how the virus exits an infected cells, remain poorly understood. The late region of the BKPyV genome encodes an auxillery protein called agnoprotein. We and others have shown that agnoprotein is an essential factor in virus release, and the loss of agnoprotein results in an accumulation of virus particles within the nucleus of an infected cell. The functions of agnoprotein necessary for this egress phenotype are not known. Here we demonstrate that agnoprotein shows properties associated with viroporins, a group of virus-encoded membrane spanning proteins that play key roles in virus infection and release. We demonstrate that agnoprotein oligomerises and perturbs membranes in cells. The development of a novel recombinant agnoprotein expression system permitted the identification of the first small molecules targeting agnoprotein. These compounds abrogated agnoprotein viroporin activity in vitro and reduced virus release, indicating that viroporin activity contributes to the phenotype observed in agnoprotein knockout viruses. The identification of channel activity should enhance the future understanding of the physiological function of agnoprotein and could represent an important target for antiviral intervention.

microbiology↗

bioRxiv2022

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain, N-terminal domain and S2 region, distal to the ACE2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target. Significance statementThis study reports on the discovery of a macrocyclic peptide that is able to inhibit SARS-CoV-2 infection by exploiting a new vulnerable site in the spike glycoprotein. This region is highly conserved across SARS-CoV-2 variants and the subgenus sarbecovirus. Due to the inaccessability and mutational contraint of this site, it is anticipated to be resistant to the development of resistance through antibody selective pressure. In addition to the discovery of a new molecule for development of potential new peptide or biomolecule therapeutics, the discovery of this broadly active conserved site can also stimulate a new direction of drug development, which together may prevent future outbreaks of related viruses.

biochemistry↗

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↗

bioRxiv2022

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity. One sentence summaryA human monoclonal antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

microbiology↗

bioRxiv2021

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes the hexameric complex

The enterovirus genus encompasses many clinically important human pathogens such as poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These viruses are the etiological agents of several human diseases, including hand-foot-and-mouth disease, neonatal sepsis, encephalitis, meningitis, paralysis and respiratory infections. There is an unmet need for antivirals to treat these diseases. The non-structural protein 2C is a AAA+ helicase and plays a key role in viral replication. As such, it is an attractive target for antiviral drug development. Several repurposing screens with FDA-approved drugs have identified 2C-targeting compounds such as fluoxetine and dibucaine, but the molecular basis of 2C inhibition has remained enigmatic. Here we present the 1.5 [A] resolution crystal structure of the soluble fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), which reveals a conserved, hydrophobic drug-binding pocket which is distal to the ATP binding site. To decipher the molecular mechanism of inhibition by fluoxetine and other 2C-targeting compounds, we engineered a soluble, hexameric and ATPase competent 2C protein. Using this system, we show that SFX, dibucaine, HBB and guanidine hydrochloride inhibit 2C ATPase activity in a dose-dependent manner. Moreover, using cryo-EM analysis, we demonstrate that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing us to generate the first reconstruction of the oligomeric complex. Taken together, these results provide important structural and mechanistic insights into 2C inhibition and provide a robust engineering strategy which can be used for structural, functional and drug-screening analysis of 2C proteins from current or future enteroviruses.

microbiology↗

bioRxiv2020

A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core

SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.

immunology↗

bioRxiv2020

Highly potent anti-SARS-CoV-2 multi-DARPin therapeutic candidates

Globally accessible therapeutics against SARS-CoV-2 are urgently needed. Here, we report the generation of the first anti-SARS-CoV-2 DARPin molecules with therapeutic potential as well as rapid large-scale production capabilities. Highly potent multivalent DARPin molecules with low picomolar virus neutralization efficacies were generated by molecular linkage of three different monovalent DARPin molecules. These multivalent DARPin molecules target various domains of the SARS-CoV-2 spike protein, thereby limiting possible viral escape. Cryo-EM analysis of individual monovalent DARPin molecules provided structural explanations for the mode of action. Analysis of the protective efficacy of one multivalent DARPin molecule in a hamster SARS-CoV-2 infection model demonstrated a significant reduction of pathogenesis. Taken together, the multivalent DARPin molecules reported here, one of which has entered clinical studies, constitute promising therapeutics against the COVID-19 pandemic.

immunology↗

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↗

bioRxiv2019

High-Resolution Cryo-EM Reveals Dynamics in the Murine Norovirus Capsid

Rather than acting as rigid symmetrical shells to protect and transmit their genomes, the capsids of non-enveloped, icosahedral viruses co-ordinate multiple, essential processes during the viral life-cycle, and undergo extensive conformational rearrangements to deliver these functions. Capturing conformational flexibility has been challenging, yet could be key in understanding and combating infections that viruses cause. Noroviruses are non-enveloped, icosahedral viruses of global importance to human health. They are a common cause of acute non-bacterial gastroenteritis, yet no vaccines or antiviral agents specific to norovirus are available. Here, we use cryo-electron microscopy to study the high-resolution solution structures of infectious, inactivated and mutant virions of murine norovirus (MNV) as a model for human noroviruses. Together with genetic studies, we show that the viral capsid is highly dynamic. While there is little change to the shell domain of the capsid, the protruding domains that radiate from this are flexible and adopt distinct states both independently and synchronously. In doing so the viral capsid is able to sample a defined range of conformational space, with implications for the maintenance of virion stability and infectivity. These data will aid in developing the first generation of effective control measures against this virus.

microbiology↗