Biology subjects

Newman, J.

Publications and source records attributed to Newman, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Characterisation of naturally occurring MERS-CoV Spike mutations and their impact on entry and neutralisation.

In this study the phenotypic consequences of naturally occurring single nucleotide polymorphisms (SNPs) in the MERS-CoV Spike protein were investigated. The impact of Spike mutations on virus entry and neutralisation of contemporary MERS-CoV strains is not currently well understood. Naturally occurring mutations were identified by aligning 584 MERS-CoV Spike sequences from either human clinical isolates collected between 2012 - 2024 or from viruses passaged in human cells. Fifteen SNPs of interest occurring in the NTD, RBD and adjacent to the S1/S2 cleavage site were selected for further characterisation based on their location in the Spike protein, frequency and identification in previous studies. A representative clade B, lineage 5 wildtype Spike sequence, which reflected those carried by MERS-CoV viruses circulating in the Middle East, was used in this study. The mutations of interest were introduced to the wildtype backbone to generate Spike variants. A lentiviral-based pseudotyping system was then used to investigate the impact of these Spike mutations on entry and neutralisation. I529T, E536K and L745F were shown to improve MERS-CoV entry. L411F, T424I, L506F, L745F and T746K were found to increase resistance to neutralisation by pooled patient sera. This study has identified novel naturally occurring Spike mutations that resulted in phenotypic differences in virus entry and neutralisation of contemporary MERS-CoV strains. Continued investigation of the phenotypic consequences of naturally occurring MERS-CoV Spike mutations is essential for assessing the risk to public health, especially given the pandemic potential of this virus. ImportanceThe main aim of this study was to investigate the impact of naturally occurring MERS-CoV Spike mutations on virus entry and neutralisation. The phenotypic consequences of mutations occurring in the Spike protein of contemporary MERS-CoV strains are not currently well understood. Improving our understanding is of particular importance due to MERS-CoV continuing to pose a public health risk, with frequent spillover events and mounting evidence of human-to-human transmission since the virus emerged in 2012. A major concern is that as MERS-CoV continues to evolve, it may become more infectious, resulting in increased transmission between humans. To add to this, surveillance is limited and there are currently no specific medical countermeasures available to treat MERS-CoV disease. The MERS-CoV Spike pseudotyping system developed in this study is a useful tool that could be used alongside surveillance systems to rapidly assess novel Spike mutations in functional assays. This MERS-CoV pseudotyping system could also be used to aid the development of medical countermeasures such as vaccines, antivirals and antibody therapies.

microbiology↗

bioRxiv2025

Tissue-wide cues are sensed at the cellular level to coordinate microtubule orientations in plants

Plant morphogenesis depends on tissue-wide coordination of cortical microtubule orientations. This coordination is thought to depend on microtubules responding to mechanical stress orientations. Here we test this stress-sensing hypothesis by quantifying microtubule behaviours on different faces and edges of growing leaf cells. We show that microtubules orientations exhibit both cell-geometric and tissue biases. Stress sensing can account for tissue biases but not microtubule trajectories and densities. An alternative hypothesis is suggested by two edge behaviours: generation of microtubules within cell edges and a filter that prevents microtubules entering side faces at shallow angles. Incorporating these features into a combinatorial model in which cell face identities modulate microtubule behaviours, accounts for tissue coordination and observed dynamics. Thus, instead of orientations being sensed at the microtubule level, they are sensed at the cellular level through changes in statistical behaviours at cell faces and edges, coordinated across a tissue through cell polarity.

plant biology↗

bioRxiv2025

Mechanisms for cross-neutralisation of diverse bat sarbecoviruses

The continuing evolution of SARS-CoV-2 variants of concern, and the increasing spillover potential of sarbecoviruses into the human population presents an important and urgent need to discover cross-reactive monoclonal antibodies (mAbs) for future therapeutic use and identify conserved neutralising epitopes that can be used for rationale design of broadly protective sarbecovirus vaccines. Here we study the neutralising epitopes on WIV-1 Spike of three mAbs that confer broad sarbecovirues and SARS-CoV-2 variant neutralisation, including XEC and JN.1. mAb V1WT_06 binds a highly conserved RBD site V epitope that is mediated by the heavy chain alone. V1WT_06 contact residues are highly conserved in circulating viruses suggesting that the epitope is evolutionarily and functionally constrained. mAbs V1WT_41 and VA14_26 bind overlapping RBD class 4 epitopes with differing angles of approach that impact on the degree of ACE2 competition. We show that neutralisation by these mAbs is maintained when virus entry is via Japanese horseshoe bat and Halcyon horseshoe bat ACE2. These mAbs are ideal candidates for therapeutic antibody development and inform the rational design of pan-coronavirus vaccines.

microbiology↗

bioRxiv2025

Understanding the future risk of bat coronavirus spillover into humans: correlating sarbecovirus receptor usage, host range, and antigenicity.

Sarbecoviruses interact with their receptor, angiotensin converting enzyme 2 (ACE2), via the receptor binding domain (RBD) of Spike, the immunodominant target for neutralising antibodies. Understanding the interplay and correlation between ACE2-determined host range and antigenicity is vitally important for understanding the zoonotic potential of related bat sarbecoviruses. Using binding assays, pseudotype-entry assays and a diverse panel of mammalian ACE2 proteins, we examined the host range and related antigenicity of multiple bat coronaviruses. Broad bat ACE2 usage (a generalist phenotype) was most common in clade 1 sarbecoviruses, including SARS-CoV-2 and the BANAL isolates from Laos. In contrast, clade 3 (e.g., RhGB07) and 5 (e.g., Rc-o319) sarbecoviruses exhibited more restricted ACE2 usage (a specialist phenotype). A novel structure for RhGB07 Spike further helped to identify RBD residues associated with this receptor specialism. Interestingly, the generalist phenotypes were largely maintained with more diverse mammalian receptor libraries, including human, non-human primate, livestock, rodent ACE2 and potential intermediate reservoir hosts (e.g., civet, racoon dog, pangolin), while specialists, like RhGB07, exhibited wider phenotypic diversity. The impact of SARS-CoV-2s continued evolution in humans was also examined, identifying an expanding and/or shifting pattern of generalism for variants, especially Omicron and its sub-lineages. Furthermore, we compared and correlated these entry phenotypes with antigenicity using sera from SARS-CoV-2 convalescent individuals. Clade 1 viruses, phylogenetically related to SARS-CoV-2, were antigenically the most similar, with robust evidence for cross-neutralisation; however, there was still evidence for limited cross-neutralisation across the entire sub-genus. Finally, using monoclonal antibodies, derived from COVID-19 vaccinees with breakthrough infections, we pin-pointed the antibody epitope classes responsible for wider neutralisation. Our research indicates that generalist ACE2-using sarbecoviruses are phylogenetically and antigenically related to SARS-CoV-2.

microbiology↗

bioRxiv2025

Metabolic Alteration in Oxylipins and Endocannabinoids Point to an Important Role for Soluble Epoxide Hydrolase and Inflammation in Alzheimer's Disease - Finding from Alzheimer's Disease Neuroimaging Initiative.

Mounting evidence implicates inflammation as a key factor in Alzheimers disease (AD) development. We previously identified pro-inflammatory soluble epoxide hydrolase (sEH) metabolites to be elevated in plasma and CSF of AD patients and to be associated with lower cognition in non-AD subjects. Soluble epoxide hydrolase is a key enzyme converting anti-inflammatory epoxy fatty acids to pro-inflammatory diols, reported to be elevated in multiple cardiometabolic disorders. Here we analyzed over 700 fasting plasma samples from the baseline of Alzheimers Disease Neuroimaging Initiative (ADNI) 2/GO study. We applied targeted mass spectrometry method to provide absolute quantifications of over 150 metabolites from oxylipin and endocannabinoids pathway, interrogating the role for inflammation/immune dysregulation and the key enzyme soluble epoxide hydrolase in AD. We provide further insights into the regulation of this pathway in different disease stages, APOE genotypes and between sexes. Additionally, we investigated in mild cognitive impaired (MCI) patients, metabolic signatures that inform about resilience to progression and conversion to AD. Key findings include I) confirmed disruption in this key central pathway of inflammation and pointed to dysregulation of sEH in AD with sex and disease stage differences; II) identified markers of disease progression and cognitive resilience using sex and ApoE genotype stratified analysis highlighting an important role for bile acids, lipid peroxidation and stress response hormone cortisol. In conclusion, we provide molecular insights into a central pathway of inflammation and links to cognitive dysfunction, suggesting novel therapeutic approaches that are based on targeting inflammation tailored for subgroups of individuals based on their sex, APOE genotype and their metabolic profile.

neuroscience↗

bioRxiv2024

Restoring the Multiple Sclerosis Associated Imbalance of Gut Indole Metabolites Promotes Remyelination and Suppresses Neuroinflammation

In multiple sclerosis (MS) the circulating metabolome is dysregulated, with indole lactate (ILA) being one of the most significantly reduced metabolites. We demonstrate that oral supplementation of ILA impacts key MS disease processes in two preclinical models. ILA reduces neuroinflammation by dampening immune cell activation as well as infiltration; and promotes remyelination and in vitro oligodendrocyte differentiation through the aryl hydrocarbon receptor (AhR). Supplementation of ILA, a reductive indole metabolite, restores the gut microbiomes oxidative/reductive metabolic balance by lowering circulating indole acetate (IAA), an oxidative indole metabolite, that blocks remyelination and oligodendrocyte maturation. The ILA-induced reduction in circulating IAA is linked to changes in IAA-producing gut microbiota taxa and pathways that are also dysregulated in MS. Notably, a lower ILA:IAA ratio correlates with worse MS outcomes. Overall, these findings identify ILA as a potential anti-inflammatory remyelinating agent and provide insights into the role of gut dysbiosis-related metabolic alterations in MS progression. One Sentence SummaryIndole lactate, a postbiotic metabolite reduced in MS, corrects gut microbiome metabolic imbalances associated with remyelination and neuroinflammation.

neuroscience↗

bioRxiv2024

Induction of neutralising antibodies against conserved rhinovirus capsid protein VP4 depends on presenting VP4 in a virus-like conformation

Infection with rhinovirus (RV) is associated with significant morbidity and hospitalisation in people with chronic lung disease. At present there is no approved RV vaccine or antiviral. There are approximately 180 RV serotypes, classified into 3 species (RVA, RVB, RVC). There is no cross-protection between serotypes because of high diversity in immunodominant antigenic sites. This makes it impractical to create a broadly protective vaccine using traditional methods, involving whole capsids. A more feasible strategy is to direct the immune response towards conserved but less dominant epitopes. The capsid protein VP4 is highly conserved within each RV genotype and some antibodies that target VP4 are neutralising. This makes VP4 vulnerable to antibodies and a promising vaccine target. Here we investigate the ability of RV VP4 N-terminal peptides presented on different display systems to initiate a neutralising immune response in mice. We compared three different-sized VP4 peptides (spanning residues, 1-15, 1-30 and 1-45) displayed on two different display systems-SpyCatcher Virus-like particles (VLPs) or Keyhole Limpet Hemocyanin (KLH). Overlapping regions of VP4 were antigenically different when presented on different platforms and in peptides with different length. The conformation of the 1-15 region of VP4 was critical for inducing antibodies that were both neutralising and able to bind VP4 in the context of the virus particle. Therefore, correctly displayed VP4 peptides can recapitulate virus-like antigenicity. These findings improve our understanding of VP4 antigenicity and will inform the design of future RV vaccines. This work could also impact the design of other peptide vaccines, since a variable antigenic conformation is a common characteristic of pathogen-derived peptide targets. Impact statementRhinovirus (RV) is a highly prevalent respiratory virus that is a frequent cause of common cold symptoms. For people with chronic lung diseases such as asthma, infection can cause significant worsening of disease, leading to increased suffering and hospitalisation. There are currently no vaccines or anti-viral drugs that target RV infections. Development of a RV vaccine would be hugely beneficial to suffers of chronic lung diseases. Attempts to develop a broadly protective RV vaccine have been hindered by the presence of over 180 serotypes which have little or no natural cross-reactivity. Previous studies have identified the highly conserved VP4 epitope as a potential target for broadly protective vaccines. However, there has been little progress in evaluating its effectiveness as a vaccine candidate. In this study we assessed the ability of different RV VP4 immunogens to induce neutralising antibodies in mice. We demonstrated that antigenic conformation of VP4 varies greatly between immunogens and presentation of the first 15 N-terminal residues in a virus-like conformation is important for generating neutralising antibodies. This knowledge could help to maximise the production of neutralising VP4-specific antibodies in future studies which could ultimately lead to a universal RV vaccine.

microbiology↗

bioRxiv2024

The pseudoknot region and poly-(C) tract comprise an essential RNA packaging signal for assembly of foot-and-mouth disease virus

Virus assembly is a crucial step for the completion of the viral replication cycle. In addition to ensuring efficient incorporation of viral genomes into nascent virions, high specificity is required to prevent incorporation of host nucleic acids. For picornaviruses, including FMDV, the mechanisms required to fulfil these requirements are not well understood. However, recent evidence has suggested that specific RNA sequences dispersed throughout picornavirus genomes are involved in packaging. Here, we have shown that such sequences are essential for FMDV RNA packaging and have demonstrated roles for both the pseudoknot (PK) region and the poly-(C) tract in this process, where the length of the poly-(C) tract was found to influence the efficiency of RNA encapsidation. Sub-genomic replicons containing longer poly-(C) tracts were packaged with greater efficiency in trans, and viruses recovered from transcripts containing short poly-(C) tracts were found to have greatly extended poly-(C) tracts after only a single passage in cells, suggesting that maintaining a long poly-(C) tract provides a selective advantage. We also characterised a critical packaging signal (PS) located in the pseudoknot (PK) region, adjacent to the poly-(C) tract, as well as several other non-essential but beneficial PSs elsewhere in the genome. Collectively, these PSs greatly enhanced encapsidation efficiency, with the poly-(C) tract possibly facilitating nearby PSs to adopt the correct conformation. Using these data, we have proposed a model where interactions with capsid precursors control a transition between two RNA conformations, directing the fate of nascent genomes to either be packaged or alternatively to act as templates for replication and/or for protein translation. Author summaryGenome packaging, whereby viral RNA is incorporated into protective protein capsids to produce more virus particles, is a crucial step in RNA virus life cycles. It is a stringent process as only viral RNA is encapsidated, while cellular RNA is excluded. This study reveals the essential role of packaging signals in FMDV RNA packaging, specifically those in the pseudoknot region and in a region that can contain >100 cytosines, termed the poly-(C) tract. We demonstrate that the length of the poly-(C) tract significantly affects packaging efficiency; genomes containing longer poly-(C) tracts are favoured. This is the first role that has been identified for the poly-(C) tract in FMDV. We have also found an essential packaging signal in the pseudoknot region, which is assisted by other packaging signals located throughout the genome, that together facilitate encapsidation of FMDV RNA. These results provide compelling evidence for the involvement of RNA packaging signals in FMDV assembly. Based on this, we propose a simple model for FMDV RNA packaging, which involves a transition from genome replication to genome packaging and is controlled by packaging signals. This knowledge could pave the way for future research and development of novel antiviral strategies targeting FMDV and other picornaviruses.

microbiology↗

bioRxiv2024

Global variation in prior exposure shapes antibody neutralization profiles of SARS-CoV-2 variants up to BA.2.86

The highly mutated SARS-CoV-2 variant, BA.2.86, and its descendants are now the most frequently sequenced variants of SARS-CoV-2. We analyze antibody neutralization data from eight laboratories from the UK, USA, Denmark, and China, including two datasets assessing the effect of XBB.1.5 vaccines, to determine the effect of infection and vaccination history on neutralization of variants up to and including BA.2.86, and produce antibody landscapes to describe these neutralization profiles. We find evidence for lower levels of immune imprinting on pre-Omicron variants in sera collected from Denmark and China, which may be explained by lower levels of circulation of the ancestral variant in these countries, and the use of an inactivated virus vaccine in China.

immunology↗

bioRxiv2023

Vgamma9Vdelta2 T cells recognize butyrophilin 2A1 and 3A1 heteromers

Butyrophilin (BTN) molecules are emerging as key regulators of T cell immunity, however, how they trigger cell-mediated responses is poorly understood. Here, the crystal structure of a gamma-delta T cell receptor ({gamma}{delta}TCR) in complex with BTN member 2A1 (BTN2A1) revealed that BTN2A1 engages the side of the {gamma}{delta}TCR, leaving the apical TCR surface bioavailable. We reveal that BTN3A1 is a second {gamma}{delta}TCR ligand, that co-engages {gamma}{delta}TCR via binding to this accessible apical surface. BTN2A1 and BTN3A1 also directly interact with each other in cis, and structural analysis revealed formation of W-shaped heteromeric multimers. This BTN2A1-BTN3A1 interaction involved the same epitopes that BTN2A1 and BTN3A1 each use to engage {gamma}{delta}TCR; indeed, either forced separation or locking together of BTN2A1 and BTN3A1 resulted in enhanced or abrogated {gamma}{delta}TCR interaction, respectively. Our findings reveal a new paradigm in immune activation, whereby {gamma}{delta}TCRs recognize dual epitopes on BTN2A1 and BTN3A1 complexes.

immunology↗

bioRxiv2023

Pixel-wise programmability enables dynamic high-SNR cameras for high-speed microscopy

High-speed wide-field fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio at high frame rates, limiting their ability to detect faint fluorescent events. Here, we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high signal-to-noise ratio. In high-speed voltage imaging experiments, our image sensor significantly increases the output signal-to-noise ratio compared to a low-noise scientific CMOS camera ([~]2-3 folds). This signal-to-noise ratio gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions.

neuroscience↗

bioRxiv2022

Combination therapy of glycation lowering compounds reduces caloric intake, improves insulin sensitivity and extends lifespan.

Non-enzymatic reactions in glycolysis lead to the accumulation of methylglyoxal (MGO), a reactive precursor to advanced glycation end-products (AGEs), which has been hypothesized to drive obesity, diabetes and aging-associated pathologies. A combination of nicotinamide, -lipoic acid, thiamine, pyridoxamine, and piperine (Gly-Low) lowered deleterious effects of glycation by reducing MGO and MGO-derived AGE, MG-H1, in mice. Gly-Low supplementation in the diet reduced food consumption, decreased body weight, improved insulin sensitivity, and increased survival in leptin receptor-deficient (Leprdb) and wild-type C57B6/J mice. Transcriptional, protein, and functional analyses demonstrated that Gly-Low inhibited appetite-stimulating ghrelin signaling and enhanced the appetite-satiating mTOR pathways within the hypothalamus. Consistent with these molecular findings, Gly-Low inhibited ghrelin-mediated hunger responses. When administered as a late-life intervention, Gly-Low slowed hypothalamic aging signatures, improved glucose homeostasis and motor coordination, and increased lifespan, suggesting its potential benefits in ameliorating age-associated decline. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=171 HEIGHT=200 SRC="FIGDIR/small/503411v3_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1beedc4org.highwire.dtl.DTLVardef@1ec1933org.highwire.dtl.DTLVardef@16a7ad2org.highwire.dtl.DTLVardef@1a5a608_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Gluebodies improve crystal reliability and diversity through transferable nanobody mutations that introduce constitutive crystal contacts

The design of proteins that may assemble in a manner that is transferable and modular remains an enduring challenge. In particular, obtaining well-diffracting protein crystals suitable for characterizing ligands or drug candidates and understanding different protein conformations remains a bottleneck for structural studies. Using nanobodies as crystallization chaperones is one strategy to address the problem, but its reliability is uncharacterized and, in this study, we observed it to have a limited success rate. Here we show that by exploring and testing the nanobody-nanobody interfaces predominant in >200 combinations of surface mutations in multiple iterations we can engineer robust crystallization behaviour into the nanobody scaffold. Strikingly, this survey yielded multiple polymorphs, all mediated by the same interface. The resulting Gluebodies (Gbs) provide far superior resolution and reliability of diffraction and can be routinely generated for chaperone experiments. We furthermore show that Gbs cannot rescue intrinsically non-crystallizing proteins, but instead are a powerful approach to improve the packing and resolution limit of poorly diffracting crystals. The discovery of an engineered, preferred nanobody interface that arises under kinetic control - trapped here by irreversible crystallization - embodies a protein assembly strategy that could prove even more broadly useful for modular assembly trapped by other irreversible methods.

biochemistry↗

bioRxiv2022

Single-nucleus RNA-seq reveals disrupted cell maturation by chorioamnionitis in the preterm cerebellum of nonhuman primates

Preterm birth is often associated with chorioamnionitis and increased risk of neurodevelopmental disorders. Preterm birth also leads to cerebellar underdevelopment but the mechanisms of disrupted cerebellar development in preterm infants are little understood. Here, we leveraged single-nuclei RNA-sequencing of the cerebellum in a rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation. Chorioamnionitis also accelerated pre-oligodendrocyte maturation into myelinating oligodendrocytes, confirmed by increased expression of myelin basic protein in the cerebellum of chorioamnionitis-exposed fetuses. These findings are consistent with reported histopathological findings in individuals with autism and suggest a novel mechanism through which perinatal inflammation contributes to neurodevelopmental disorders.

neuroscience↗

bioRxiv2022

Distinct antigenic properties of the SARS-CoV-2 Omicron lineages BA.4 and BA.5

Over the course of the pandemic variants have arisen at a steady rate. The most recent variants to emerge, BA.4 and BA.5, form part of the Omicron lineage and were first found in Southern Africa where they are driving the current wave of infection. In this report, we perform an in-depth characterisation of the antigenicity of the BA.4/BA.5 Spike protein by comparing sera collected post-vaccination, post-BA.1 or BA.2 infection, or post breakthrough infection of vaccinated individuals with the Omicron variant. In addition, we assess sensitivity to neutralisation by commonly used therapeutic monoclonal antibodies. We find sera collected post-vaccination have a similar ability to neutralise BA.1, BA.2 and BA.4/BA.5. In contrast, in the absence of vaccination, prior infection with BA.2 or, in particular, BA.1 results in an antibody response that neutralises BA.4/BA.5 poorly. Breakthrough infection with Omicron in vaccinees leads to a broad neutralising response against the new variants. The sensitivity of BA.4/BA.5 to neutralisation by therapeutic monoclonal antibodies was similar to that of BA.2. These data suggest BA.4/BA.5 are antigenically distinct from BA.1 and, to a lesser extent, BA.2. The enhanced breadth of neutralisation observed following breakthrough infection with Omicron suggests that vaccination with heterologous or multivalent antigens may represent viable strategies for the development of cross-neutralising antibody responses.

microbiology↗

bioRxiv2022

An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies have uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 entry and fusion independent of transmembrane protease serine 2 expression in multiple cell types. We also demonstrate a role for ACAT in regulating SARS-CoV-2 RNA replication in primary bronchial epithelial cells. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled in the acute phase of infection. Thus, re-purposing of available ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.

immunology↗

bioRxiv2022

Crystal structures and fragment screening of SARS-CoV-2 NSP14 reveal details of exoribonuclease activation and mRNA capping and provide starting points for antiviral drug development

The SARS-CoV-2 non-structural protein 14 (NSP14) is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both enzymatic activities appear to be essential for the viral life cycle and thus may be targeted for anti-viral therapeutics. NSP14 forms a stable complex with the SARS-CoV-2 zinc binding protein NSP10, and this interaction greatly enhances the nuclease but not the methyltransferase activity. In this study, we have determined the crystal structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 [A] resolution. Comparisons of this structure with the structure of NSP14/NSP10 complexes solved to date reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including significant movements and helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. Conformational changes are also seen in the MTase active site within a SAM/SAH interacting loop that plays a key role in viral mRNA capping. We have also determined the structure of NSP14 in complex with cap analogue 7MeGpppG, offering new insights into MTase enzymatic activity. We have used our high resolution crystals to perform X-ray fragment screening of NSP14, revealing 72 hits bound to potential sites of inhibition of the ExoN and MTase domains. These structures serve as excellent starting point tools for structure guided development and optimization of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.

biochemistry↗

bioRxiv2022

Membrane interactions and uncoating of Aichi virus, a picornavirus that lacks a VP4

Kobuviruses are an unusual and poorly characterised genus within the picornavirus family, and can cause gastrointestinal enteric disease in humans, livestock and pets. The human Kobuvirus, Aichi virus (AiV) can cause severe gastroenteritis and deaths in children below the age of five years, however this is a very rare occurrence. During the assembly of most picornaviruses (e.g. poliovirus, rhinovirus and foot-and-mouth disease virus), the capsid precursor protein VP0 is cleaved into VP4 and VP2. However, Kobuviruses retain an uncleaved VP0. From studies with other picornaviruses, it is known that VP4 performs the essential function of pore formation in membranes, which facilitates transfer of the viral genome across the endosomal membrane and into the cytoplasm for replication. Here, we employ genome exposure and membrane interaction assays to demonstrate that pH plays a critical role in AiV uncoating and membrane interactions. We demonstrate that incubation at low pH alters the exposure of hydrophobic residues within the capsid, enhances genome exposure and enhances permeabilisation of model membranes. Furthermore, using peptides we demonstrate that the N-terminus of VP0 mediates membrane pore formation in model membranes, indicating that this plays an analogous function to VP4. ImportanceTo initiate infection, viruses must enter a host cell and deliver their genome into the appropriate location. The picornavirus family of small non-enveloped RNA viruses includes significant human and animal pathogens and are also models to understand the process of cell entry. Most picornavirus capsids contain the internal protein VP4, generated from cleavage of a VP0 precursor. During entry, VP4 is released from the capsid. In enteroviruses this forms a membrane pore, which facilitates genome release into the cytoplasm. Due to high levels of sequence similarity, it is expected to play the same role for other picornaviruses. Some picornaviruses, such as Aichi virus, retain an intact VP0, and it is unknown how these viruses re-arrange their capsids and induce membrane permeability in the absence of VP4. Here we have used Aichi virus as a model VP0 virus to test for conservation of function between VP0 and VP4. This could enhance understanding of pore function and lead to development of novel therapeutic agents that block entry.

microbiology↗