Biology subjects

van Kuppeveld, F. J. M.

Publications and source records attributed to van Kuppeveld, F. J. M..

At least 19 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

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

Allosteric Disordering of eIF2B Regulates the Integrated Stress Response

The ternary complex (TC), composed of translation initiation factor eIF2, GTP, and initiator methionyl tRNA, delivers the first amino acid to the ribosome to initiate protein synthesis. The activity of the decameric eukaryotic initiation factor 2B complex (eIF2B) initiates TC assembly by catalyzing GDP to GTP exchange on eIF2, thereby setting the TC levels in the cell. Stress-induced phosphorylation converts eIF2 from the substrate of the GDP/GTP exchange reaction into an inhibitor (eIF2-P) of eIF2B. This conversion reduces the cells TC levels and induces the widespread reprogramming of translation known as the Integrated Stress Response (ISR). Here, we chart an allosteric axis running through eIF2B, revealing the importance of a protrusive -helix in its {beta}-subunit, the latch-helix, that locks onto the -subunit to induce eIF2B activity. eIF2-P binding unhooks the latch-helix, opening eIF2B, which inhibits its GDP/GTP exchange activity. Distinct viral proteins have convergently evolved to bind to eIF2B and stabilize the latch-helix-bound active state. Using these insights, we generated ISR-ACTivating compounds, ISRACTs, that stabilize eIF2B in its inhibited, unlatched state. Our study thus highlights how state-transitions in eIF2B are regulated via long-range allostery.

biochemistry↗

bioRxiv2025

Cardiovirus-Mediated PKR Inhibition Results from Nucleocytoplasmic Trafficking Disruption

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), know as PKR, is a key antiviral kinase activated by double-stranded RNA (dsRNA) typically produced during viral replication. Upon activation, PKR phosphorylates eIF2, leading to the inhibition of translation and viral replication. However, many viruses have evolved mechanisms to counteract PKR activity. In Cardioviruses, the Leader protein (L), a short peptide cleaved from the N-terminus of the viral polyprotein, not only inhibits PKR but also blocks interferon production and disrupts nucleocytoplasmic trafficking (NCT). L disrupts NCT by recruiting host RSK kinases to the nuclear pore complex (NPC), where RSK phosphorylates FG-nucleoporins, thereby impairing NCT. L mutations that affect NCT disruption also impact its ability to inhibit PKR, suggesting a mechanistic link. Recombinant TMEV and EMCV viruses designed to disrupt NCT through different mechanisms exhibited some extent of PKR inhibition, supporting the link between NCT disruption and PKR inhibition. Immunostaining and live-cell imaging revealed that L-induced NCT disruption redistributes a fraction of PKR to the nucleoli, where PKR remains inactive. This suggests that nucleolar sequestration contributes to PKR inhibition. Additionally, L-mediated NCT disruption leads to the release of nuclear RNA-binding proteins (nRBPs) into the cytosol, which may bind or modify viral dsRNA, further preventing PKR activation. Collectively, these results highlight nucleocytoplasmic trafficking as a critical regulatory mechanism governing PKR activation. Thus, beyond the specific action of cardiovirus L protein, our study reveals that interference with host nucleocytoplasmic transport can significantly impact the subcellular localization and functional regulation of immune effectors such as PKR. Author SummaryProtein kinase R (PKR) is a crucial component of the host innate immune response. It is activated by double-stranded RNA (dsRNA) typically produced during viral replication and triggers a shutdown of mRNA translation. This antiviral mechanism limits viral propagation by inhibiting both host and viral protein synthesis. However, many viruses have developed mechanisms to inhibit PKR, allowing them to escape immune detection. PKR downregulation facilitates viral replication whereas uncontrolled PKR activation can lead to autoimmune disorders. Therefore, PKR activity must be tightly regulated to maintain immune homeostasis. Using recombinant viruses which target the nuclear pore complex, we show that nucleocytoplasmic trafficking of cellular components is critical for regulation of PKR activity. Infection of cells with Theilers murine encephalomyelitis virus triggers an efflux of nuclear RNA binding proteins which likely compete with PKR for dsRNA binding and thereby block PKR activity. Moreover, upon TMEV infection as well as during mitosis, PKR is detected in the nucleoli where it is thought to interact with structured RNAs without being activated. Our data highlight an important link between nucleocytoplasmic trafficking and PKR activity.

immunology↗

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↗

bioRxiv2025

Identification of picornavirus proteins that inhibit de novo nucleotide synthesis during infection

The picornavirus family includes well-known pathogens for humans and animals, such as enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) and cardioviruses (e.g. encephalomyocarditis virus [EMCV] and Saffold virus). Picornaviruses modulate cellular metabolism likely to generate sufficient building blocks for virus replication. Previously, we showed that coxsackievirus B3 (CVB3) and EMCV remodel nucleotide metabolism during infection. Here, we investigated whether this modulation is attributable to specific viral proteins. For this, we studied the modulation of metabolism by several recombinant CVB3 and EMCV viruses in HeLa cells. Using isotope tracing metabolomics with three distinct labels, 13C6-glucose or 13C5/15N2-glutamine, we reveal that the 2A protease of CVB3 and the Leader protein of EMCV inhibit de novo nucleotide synthesis. Furthermore, we show that nucleotide metabolism is also reprogrammed by CVB3 and EMCV in human induced pluripotent stem cell-derived cardiomyocytes. Our insights are important to increase understanding of picornavirus-host interactions and may lead to novel therapeutic strategies.

microbiology↗

bioRxiv2025

ITAF45 is a Pervasive Trans Acting Factor for Picornavirus Type II IRES Elements

Viruses have evolved elaborate mechanisms to hijack the host mRNA translation machinery to direct viral protein synthesis. Picornaviruses, whose RNA genomes lack a cap structure, inhibit cap-dependent mRNA translation, and utilize an internal ribosome entry site (IRES) in the RNA 5'-UTR to recruit the 40S ribosomal subunit. IRES activity is stimulated by a set of host proteins termed IRES trans-acting factors (ITAFs). The cellular protein ITAF45 (also known as PA2G4 and EBP1) was identified as an essential ITAF for foot-and-mouth disease virus (FMDV), with no apparent role in cell-free systems for the closely related viruses harboring similar IRES elements such as encephalomyocarditis virus (EMCV) and Theilers murine encephalomyelitis virus (TMEV). Here, we demonstrate that ITAF45 is a pervasive host factor within cells for picornaviruses containing a Type II IRES. CRISPR/Cas9 knockout of ITAF45 in several human cell lines conferred resistance to infection with FMDV, EMCV, TMEV, and equine rhinitis A virus (ERAV). We show that ITAF45 enhances initiation of translation on type II IRESs in cell line models. This is mediated by the C-terminal lysine-rich region of ITAF45 known to enable binding to viral RNA. These findings challenge previous reports of a unique role for ITAF45 in FMDV infection, positioning ITAF45 as a promising antiviral target for various animal viruses and emerging human cardioviruses.

microbiology↗

bioRxiv2025

The critical role of enterovirus 2A protease in viral translation, replication, and antagonism of host antiviral responses.

Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. Proteases 2Apro and 3Cpro have been implicated in these processes based on in vitro studies, ectopic overexpression, and surrogate infection systems, but their relative contributions are unknown. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2Apro. Viruses with an inactive 2Apro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2Apro, but not 3Cpro, is essential during infection for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, cleaving retinoic acid-inducible gene-1 (RIG-I)-like receptor (RLR) signaling pathway proteins and suppressing interferon-/{beta} (IFN-/{beta}) transcription, and overcoming the antiviral action of IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2Apro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. In conclusion, we establish that 2Apro plays a critical role in subverting antiviral responses and establishing a favorable host environment to expedite enterovirus replication.

microbiology↗

bioRxiv2024

Molecular basis of autoimmune disease protection by MDA5 variants

MDA5 recognizes dsRNA from viruses and retroelements. Cooperative filament formation and ATP-dependent proofreading confer MDA5 with the necessary sensitivity and specificity for dsRNA. The gene encoding MDA5 is a hotspot for disease-associated variants. Many MDA5 variants are associated with protection from autoimmune disease, while increasing the risk of infection and chronic inflammation, but how these variants affect MDA5-dependent RNA sensing remains unclear. Here, we determine the consequences of autoimmune-protective MDA5 variants on the molecular structure and activities of MDA5. The rare variants E627* and I923V reduce the cellular interferon response to picornavirus infection and are deficient in filament formation. The I923V variant is ATPase hyperactive, causing premature dissociation from dsRNA. Cryo-EM structures of MDA5 I923V bound to dsRNA at different stages of ATP hydrolysis reveal smaller RNA binding interfaces, leading to excessive proofreading activity. Variants R843H and T946A, which are genetically linked and cause mild phenotypes, have no effect on dsRNA recognition, suggesting an indirect disease mechanism. We conclude that the autoimmune-protective MDA5 variants lead to a loss of MDA5-dependent signaling via multiple distinct mechanisms.

biochemistry↗

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

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

Heterogeneity in viral replication dynamics shapes the antiviral response

In response to virus infection, host cells can activate antiviral signaling to restrict virus replication and communicate viral infection to neighboring cells. For poorly understood reasons, antiviral response activation is highly heterogeneous among infected cells; both quantitatively (level of pathway activation) and qualitatively (transcribed antiviral gene set). Here, we used live-cell single-molecule imaging to simultaneously visualize viral infection and antiviral signaling, providing quantitative insights into antiviral response activation in single cells; first, the probability of activating an antiviral response varies throughout infection, with most efficient activation occurring several hours after the first viral replication. Second, cell-to-cell heterogeneity in viral replication rates early in infection determine the efficiency of antiviral response activation. Finally, variation in signaling strength of the viral sensing pathway result in qualitatively distinct antiviral responses. Together, this works identifies key parameters that shape the antiviral response and provides quantitative insights into the origin of heterogeneity in the antiviral response.

immunology↗

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

The encephalomyocarditis virus Leader promotes the release of virions inside extracellular vesicles via the induction of secretory autophagy

Naked viruses can escape host cells before the induction of lysis via release in extracellular vesicles (EVs). These nanosized EVs cloak the secreted virus particles in a host-derived membrane, which alters virus-host interactions that affect infection efficiency and antiviral immunity. Currently, little is known about the viral and host factors regulating this form of virus release. Here, we assessed the role of picornavirus security proteins by studying the encephalomyocarditis virus (EMCV) Leader protein. EV release upon infection with wildtype virus or a Leader-deficient mutant was characterized at the single particle level using high-resolution flow cytometry. Inactivation of the Leader abolished EV induction during infection and strongly reduced EV-enclosed virus release. We demonstrate that the Leader promotes the release of virions within EVs by stimulating a secretory arm of autophagy. This newly discovered role of the EMCV Leader adds to the variety of mechanisms via which this protein affects virus-host interactions. Moreover, these data provide first evidence for a crucial role of non-structural viral proteins in the non-lytic release of picornaviruses via packaging in EVs.

cell biology↗

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↗

bioRxiv2021

An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces sterile immunity and prevents transmission of SARS-CoV-2 between cats

Early in the global SARS-CoV-2 pandemic concerns were raised regarding infection of other animal hosts and whether these could play a significant role in the viral epidemiology. Infection of animals could be detrimental by causing clinical disease but also of concern if they become a viral reservoir allowing further mutations, plus having the potential to infect other animals or humans. The first reported animals to be infected both under experimental conditions and from anecdotal field evidence were cats described in China early in 2020. Given the concerns this finding raised and the close contacts between humans and cats, we aimed to determine whether a vaccine candidate could be developed that was suitable for use in multiple susceptible animal species and whether this vaccine could reduce infection of cats in addition to preventing spread to other cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus (VEEV), known to be safe and efficacious for use in a variety of animals, expressing a stabilised Spike antigen, could induce neutralising antibody titers in guinea pigs and cats. After two intramuscular vaccinations, virus neutralising antibodies were detected in the respiratory tract of the guinea pigs and a cell mediated immune response was induced. The design of the SARS-CoV-2 antigen was shown to be critical in developing a strong neutralising antibody response. Vaccination of cats was able to induce a serum neutralising antibody response which lasted for the course of the experiment. Interestingly, in contrast to control animals, infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after challenge. Moreover, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit virus. The results show that the RP vaccine induces sterile immunity preventing SARS-CoV-2 infection and transmission. This data suggests that this RP vaccine could be a multi-species vaccine useful for preventing spread to and between other animals should that approach be required.

microbiology↗