ABSTRACT
Since December 2019 the SARS-CoV-2 virus has infected billions of people around the world and caused millions of deaths. The ability for this RNA virus to mutate has produced variants that have been responsible for waves of infections across the globe. The spike protein on the surface of the SARS-CoV-2 virion is responsible for cell entry in the infection process. Here we have studied the spike proteins from the Original, Alpha (B.1.1.7), Delta (B1.617.2), Delta-plus (B1.617.2-AY1), Omicron BA.1 and Omicron BA.2 variants. Using models built from cryo-EM structures with linoleate bound (6BZ5.pdb) and the N-terminal domain from 7JJI.pdb, each is built from the first residue, with missing loops modelled and 45 disulphides per trimer. Each spike variant was modified from the same Original model framework to maximise comparability. Three replicate, 200 ns atomistic molecular dynamics simulations were performed for each case. (These data also provide the basis for further, non-equilibrium molecular dynamics simulations, published elsewhere.) The analysis of our equilibrium molecular dynamics reveals that sequence variation at the closed receptor binding domain interface particularly for Omicron BA.2 has implications for the avidity of the locked conformation, with potential effects on Omicron BA.1 and Delta-plus. Linoleate binding has a mildly stabilizing effect on furin cleavage site motions in the Original and Alpha variants, but has no effect in Delta, Delta-plus and slightly increases motions at this site for Omicron BA.1, but not BA.2, under these simulation conditions.
ABSTRACT
Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilised pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID- 19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in- house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterised samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.
ABSTRACT
As COVID-19 persists, severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor-binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA-binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic {beta}-coronaviruses ({beta}-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2 and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation while the open, infectious conformation is LA-free. Electron tomography of SARS-CoV-2 infected cells reveals that LA-treatment inhibits viral replication, resulting in fewer, deformed virions. Our results establish FFA-binding as a hallmark of pathogenic {beta}-CoV infection and replication, highlighting potential antiviral strategies. One-Sentence SummaryFree fatty acid-binding is conserved in pathogenic {beta}-coronavirus S proteins and suppresses viral infection and replication.
ABSTRACT
The SARS-CoV-2 spike protein contains a fatty acid binding site, also found in some other coronaviruses (e.g. SARS-CoV), which binds linoleic acid and is functionally important. When occupied by linoleic acid, it reduces infectivity, by locking the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the response of spike variants to linoleic acid removal. These simulations show that the fatty acid site is coupled to functional regions of the protein, some of them far from the site (e.g. in the receptor-binding motif, N-terminal domain, the furin cleavage site located in position 679-685 and the fusion peptide-surrounding regions) and identify the allosteric networks involved in these connections. Comparison of the response of the original ( Wuhan) spike with four variants: Alpha, Delta, Delta plus and Omicron BA.1 show that the variants differ significantly in their response to linoleic acid removal. The allosteric connections to the fatty acid site on Alpha are generally similar to the original protein, except for the receptor-binding motif and S71-R78 region which show a weaker link to the FA site. In contrast, Omicron is the most affected variant exhibiting significant differences in the receptor-binding motif, N-terminal domain, V622-L629 and the furin cleavage site. These differences in allosteric modulation may be of functional relevance, e.g. in differences in transmissibility and virulence. Experimental comparison of the effects of linoleic acid on different variants is warranted.
ABSTRACT
Saliva is easily obtainable non-invasively and potentially suitable for detecting both current and previous SARS-CoV-2 infection. We established 6 standardised enzyme linked immunosorbent assays (ELISA) capable of detecting IgA and IgG antibodies to whole SARS-CoV-2 spike protein, to its receptor binding domain region and to nucleocapsid protein in saliva. In test accuracy (n=320), we found that spike IgG performed best (ROC AUC: 95.0%, 92.8-97.3%), followed by spike IgA (ROC AUC: 89.9%, 86.5-93.2%) for discriminating between pre-pandemic and post COVID-19 saliva samples. Using machine learning, diagnostic performance was improved when a combination of tests was used. As expected, salivary IgA was poorly correlated with serum, indicating an oral mucosal response whereas salivary IgG responses were predictive of those in serum. When deployed to 20 household outbreaks undergoing Delta and Omicron infection, antibody responses were heterogeneous but remained a reliable indicator of recent infection. Intriguingly, unvaccinated children showed evidence of exposure almost exclusively through specific IgA responses in the absence of evidence of viral infection. We have provided robust standardisation, evaluation, and field-testing of salivary antibody assays as tools for monitoring SARS-CoV-2 immune responses. Future work should focus on investigating salivary antibody responses following infection and vaccination to understand patterns of SARS-CoV-2 transmission and inform ongoing vaccination strategies.
ABSTRACT
The SARS-CoV-2 spike protein is the first contact point between the SARS-CoV-2 virus and host cells and mediates membrane fusion. Recently, a fatty acid binding site was identified in the spike (Toelzer et al. Science 2020). The presence of linoleic acid at this site modulates binding of the spike to the human ACE2 receptor, stabilizing a locked conformation of the protein. Here, dynamical-nonequilibrium molecular dynamics simulations reveal that this fatty acid site is coupled to functionally relevant regions of the spike, some of them far from the fatty acid binding pocket. Removal of a ligand from the fatty acid binding site significantly affects the dynamics of distant, functionally important regions of the spike, including the receptor-binding motif, furin cleavage site and fusion-peptide-adjacent regions. The results also show significant differences in behaviour between clinical variants of the spike: e.g. the D614G mutation shows a significantly different conformational response for some structural motifs relevant for binding and fusion. The simulations identify structural networks through which changes at the fatty acid binding site are transmitted within the protein. These communication networks significantly involve positions that are prone to mutation, indicating that observed genetic variation in the spike may alter its response to linoleate binding and associated allosteric communication.
ABSTRACT
As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriS{Delta} variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we elucidate the structure, function and molecular dynamics of this variant spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Moreover, our study reveals long-range allosteric communication between functional regions within the spike that differ in wild-type and deletion variant. Our results support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.
ABSTRACT
Many host pathogen interactions such as human viruses (including non-SARS-coronaviruses) rely on attachment to host cell-surface glycans. There are conflicting reports about whether the Spike protein of SARS-CoV-2 binds to sialic acid commonly found on host cell-surface N-linked glycans. In the absence of a biochemical assay, the ability to analyze the binding of glycans to heavily- modified proteins and resolve this issue is limited. Classical Saturation Transfer Difference (STD) NMR can be confounded by overlapping sugar resonances that compound with known experimental constraints. Here we present universal saturation transfer analysis (uSTA), an NMR method that builds on existing approaches to provide a general and automated workflow for studying protein-ligand interactions. uSTA reveals that B-origin-lineage-SARS-CoV-2 spike trimer binds sialoside sugars in an end on manner and modelling guided by uSTA localises binding to the spike N-terminal domain (NTD). The sialylated-polylactosamine motif is found on tetraantennary human N-linked-glycoproteins in deeper lung and may have played a role in zoonosis. Provocatively, sialic acid binding is abolished by mutations in some subsequent SARS- CoV-2 variants-of-concern. A very high resolution cryo-EM structure confirms the NTD location and end on mode; it rationalises the effect of NTD mutations and the structure-activity relationship of sialic acid analogues. uSTA is demonstrated to be a robust, rapid and quantitative tool for analysis of binding, even in the most demanding systems. Extended AbstractThe surface proteins found on both pathogens and host cells mediate entry (and exit) and influence disease progression and transmission. Both types can bear host-generated post- translational modifications such as glycosylation that are essential for function but can confound biophysical methods used for dissecting key interactions. Several human viruses (including non- SARS-coronaviruses) attach to host cell-surface N-linked glycans that include forms of sialic acid (sialosides). There remains, however, conflicting evidence as to if or how SARS-associated coronaviruses might use such a mechanism. Here, we demonstrate quantitative extension of saturation transfer protein NMR methods to a complete mathematical model of the magnetization transfer caused by interactions between protein and ligand. The method couples objective resonance-identification via a deconvolution algorithm with Bloch-McConnell analysis to enable a structural, kinetic and thermodynamic analysis of ligand binding beyond previously-perceived limits of exchange rates, concentration or system. Using an automated and openly available workflow this universal saturation transfer analysis (uSTA) can be readily-applied in a range of even heavily-modified systems in a general manner to now obtain quantitative binding interaction parameters (KD, kEx). uSTA proved critical in mapping direct interactions between natural sialoside sugar ligands and relevant virus-surface attachment glycoproteins - SARS-CoV-2-spike and influenza-H1N1-haemagglutinin variants - by quantitating ligand signal in spectral regions otherwise occluded by resonances from mobile protein glycans (that also include sialosides). In B- origin-lineage-SARS-CoV-2 spike trimer end on-binding to sialoside sugars was revealed contrasting with extended surface-binding for heparin sugar ligands; uSTA-derived constraints used in structural modelling suggested sialoside-glycan binding sites in a beta-sheet-rich region of spike N-terminal domain (NTD). Consistent with this, uSTA-glycan binding was minimally- perturbed by antibodies that neutralize the ACE2-binding domain (RBD) but strongly disrupted in spike from the B1.1.7/alpha and B1.351/beta variants-of-concern, which possess hotspot mutations in the NTD. Sialoside binding in B-origin-lineage-NTD was unequivocally pinpointed by cryo-EM to a site that is created from residues that are notably deleted in variants (e.g. H69,V70,Y145 in alpha). An analysis of beneficial genetic variances in cohorts of patients from early 2020 suggests a model in which this site in the NTD of B-origin-lineage-SARS-CoV-2 (but not in alpha/beta-variants) may have exploited a specific sialylated-polylactosamine motif found on tetraantennary human N-linked-glycoproteins in deeper lung. Together these confirm a novel binding mode mediated by the unusual NTD of SARS-CoV-2 and suggest how it may drive virulence and/or zoonosis via modulation of glycan attachment. Since cell-surface glycans are widely relevant to biology and pathology, uSTA can now provide ready, quantitative, widespread analysis of complex, host-derived and post-translationally modified proteins with putative ligands relevant to disease even in previously confounding complex systems.
ABSTRACT
Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known whether heart vascular pericytes (PCs) are infected by SARS-CoV-2, and if the S protein alone provokes PC dysfunction. Here, we aimed to investigate the effects of the S protein on primary human cardiac PC signalling and function. Results show, for the first time, that cardiac PCs are not permissive to SARS-CoV-2 infection in vitro, whilst a recombinant S protein alone elicits functional alterations in PCs. This was documented as: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors responsible for EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation and rescued PC function in the presence of the S protein. In conclusion, our findings suggest that circulating S protein prompts vascular PC dysfunction, potentially contributing to establishing microvascular injury in organs distant from the site of infection. This mechanism may have clinical and therapeutic implications. Clinical perspectiveO_LISevere COVID-19 manifests as a microvascular syndrome, but whether SARS-CoV-2 infects and damages heart vascular pericytes (PCs) remains unknown. C_LIO_LIWe provide evidence that cardiac PCs are not infected by SARS-CoV-2. Importantly, we show that the recombinant S protein alone elicits cellular signalling through the CD147 receptor in cardiac PCs, thereby inducing cell dysfunction and microvascular disruption in vitro. C_LIO_LIThis study suggests that soluble S protein can potentially propagate damage to organs distant from sites of infection, promoting microvascular injury. Blocking the CD147 receptor in patients may help protect the vasculature not only from infection, but also from the collateral damage caused by the S protein. C_LI
ABSTRACT
COVID-19, caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms driving high infectivity, broad tissue tropism and severe pathology. Our cryo-EM structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains (RBDs) tightly and specifically bind the essential free fatty acid (FFA) linoleic acid (LA) in three composite binding pockets. The pocket also appears to be present in the highly pathogenic coronaviruses SARS-CoV and MERS-CoV. Lipid metabolome remodeling is a key feature of coronavirus infection, with LA at its core. LA metabolic pathways are central to inflammation, immune modulation and membrane fluidity. Our structure directly links LA and S, setting the stage for interventions targeting LA binding and metabolic remodeling by SARS-CoV-2. One Sentence SummaryA direct structural link between SARS-CoV-2 spike and linoleic acid, a key molecule in inflammation, immune modulation and membrane fluidity.
