Allosteric modulation by the fatty acid site in the glycosylated SARS-CoV-2 spike (preprint)

The trimeric spike protein plays an essential role in the SARS-CoV-2 virus lifecycle, facilitating virus entry through binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediating viral and host membrane fusion. The SARS-CoV-2 spike contains an allosteric fatty acid (FA) binding site at the interface between two neighbouring receptor-binding domains. This site, also found in some other coronaviruses, binds free fatty acids such as linoleic and oleic acid, and other small molecules. Understanding allostery and how this site modulates the behaviour of different regions in this protein could potentiate the development of promising alternative strategies for new coronavirus therapies. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to investigate allosteric effects and identify the communication pathways in the fully glycosylated spike in the original SARS-CoV-2 ancestral variant. The results reveal the allosteric networks that connect the FA site to important functional regions of the protein, including some more than 40 [A] away. These regions include the receptor binding motif, an antigenic supersite in the N-terminal domain, the furin cleavage site, the regions surrounding the fusion peptide and a second allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected connection between different allosteric sites. Notably, 65% of amino acid substitutions, deletions and insertions in the Alpha, Beta, Delta, Gamma and Omicron variants map onto or close to the identified allosteric pathways.

Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine (preprint)

COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.

Molecular dynamics of spike variants in the locked conformation: RBD interfaces, fatty acid binding and furin cleavage sites. (preprint)

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.

Development and evaluation of low-volume tests to detect and characterise antibodies to SARS-CoV-2 (preprint)

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.

The Free Fatty Acid-Binding Pocket is a Conserved Hallmark in Pathogenic β-Coronavirus Spike Proteins from SARS-CoV to Omicron (preprint)

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.

SARS-CoV-2 spike variants differ in their allosteric response to linoleic acid (preprint)

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.

Evaluation of isotype specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults (preprint)

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.

The fatty acid site is coupled to functional motifs in the SARS-CoV-2 spike protein and modulates spike allosteric behaviour (preprint)

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.

Structure and mechanism of SARS-CoV-2 Spike N679-V687 deletion variant elucidate cell-type specific evolution of viral fitness (preprint)

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 analyzed 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, allowing the virus to probe diverse trajectories in distinct cell types to evolve viral fitness. TeaserSARS-CoV-2 can exploit different cell types to diversify and evolve virus variants distinct in infectivity and structure.

Changes in SARS-CoV-2 before the peak in each country, producing unique variants. (preprint)

The second and third waves of coronavirus disease 2019 (COVID-19) have caused problems worldwide. Those are often thought to have resulted from people's carelessness or people not following restrictions, but in reality, the cause remains unclear. Here, using an objective analytical method, we present the changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19 over time. The virus has mutated in three major directions, with three groups remaining to date. The basic structure of the groups was completed by April and shared across all continents. However, the virus continued to mutate independently in each country after the borders were closed. In particular, the virus mutated before the occurrence of a second or third peak. It seems that the mutations conferred higher infectivity to the virus, because of which the virus overcame previously effective protections. Currently, each country may possess such a unique stronger variant, which may cause another peak in other countries. These viruses could also serve as sources of mutations by exchanging parts of the genome. Such mutations could create a variant with superior infectivity.

The SARS-CoV-2 spike protein disrupts the cooperative function of human cardiac pericytes - endothelial cells through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease (preprint)

Background: Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses primarily the capsid spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known if the S protein alone, without the other viral elements, is able to trigger vascular cell signalling and provoke cell dysfunction. Methods: We investigated the effects of the recombinant, stabilised S protein on primary human cardiac pericytes (PCs) signalling and function. Endpoints included cell viability, proliferation, migration, cooperation with endothelial cells (ECs) in angiogenesis assays, and release of pro-inflammatory cytokines. Adopting a blocking strategy against the S protein receptors ACE2 and CD147, we explored which receptor mediates the S protein signalling in PCs. Findings: We show, for the first time, that the recombinant S protein alone elicits functional alterations in cardiac PCs. This was documented as: (1) increased migration, (2) reduced ability to support 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. Furthermore, 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 cardiac PCs. Accordingly, the neutralization of CD147, using a blocking antibody, prevented the activation of ERK1/2 and partially rescued the PC function in the presence of the S protein. Interpretation: Our findings suggest the new, intriguing hypothesis that the S protein may elicit vascular cell dysfunction, potentially amplifying, or perpetuating, the damage caused by the whole coronavirus. This mechanism may have clinical and therapeutic implication.

In vitro characterization of engineered red blood cells as potent viral traps against HIV-1 and SARS-CoV-2 (preprint)

Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps as RBCs lack nuclei and other organelles required for viral replication. Here we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein on enucleated RBCs. Engineered RBCs expressing CD4 and CCR5 were efficiently infected by HIV-1, but CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 in vitro. To facilitate continuous large-scale production of engineered RBCs, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against viral infections.

pH and Receptor Induced Confirmational Changes- Implications Towards S1 Dissociation of SARS-CoV2 Spike Glycoprotein (preprint)

Viruses, being obligate intracellular parasites, must first attach themselves and gain entry into host cells. Viral fusion machinery is the central player in the viral attachment process in almost every viral disease. Viruses have incorporated an array of efficient fusion proteins on their surfaces to bind efficiently to host cell receptors. They make use of the host proteolytic enzymes to rearrange their surface protein(s) into the form which facilitates their binding to host-cell membrane proteins and subsequently, fusion. This stage of viral entry is very critical and has many therapeutic implications. The current global pandemic of COVID-19 has sparked severe health crisis and economic shutdowns. SARS-CoV2, the etiological agent of the disease has led to millions of deaths and brought the scientific community together in an attempt to understand the mechanisms of SARS-CoV2 pathogenesis and mortality. Like other viral fusion machinery, CoV2 spike (S) glycoprotein- 'The Demogorgon' poses the same questions about viral-host cell fusion. The intermediate stages of S protein-mediated viral fusion are unclear owing to the lack of structural insights and concrete biochemical evidence. The mechanism of conformational transition is still unclear. S protein binding and fusion with host cell receptors, Eg., angiotensin-converting enzyme-2 (ACE2) is accompanied by cleavage of S1/S2 subunits. To track the key events of viral-host cell fusion, we have identified (in silico) that low pH-induced conformational change and ACE-2 binding events promote S1 dissociation. Deciphering key mechanistic insights of SARS-CoV2 fusion will further our understanding of other class- I fusion proteins.

Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands binding the Free Fatty Acid Pocket of SARS-CoV-2 Spike Protein (preprint)

Following our recent identification of a fatty acid binding site in the SARS-CoV-2 spike protein (Toelzer et al., Science eabd3255 (2020)), we investigate the binding of linoleate and other potential ligands at this site using molecular dynamics simulations. The results support the hypothesis that linoleate stabilises the locked form of the spike, in which its interaction interface for the ACE2 receptor is occluded. The simulations indicate weaker binding of linoleate to the partially open conformation. Simulations of dexamethasone bound at this site indicate that it binds similarly to linoleate, and thus may also stabilize a locked spike conformation. In contrast, simulations suggest that cholesterol bound at this site may destabilize the locked conformation, and in the open conformation, may preferentially bind at an alternative site in the hinge region between the receptor binding domain and the domain below, which could have functional relevance. We also use molecular docking to identify potential ligands that may bind at the fatty acid binding site, using the Bristol University Docking Engine (BUDE). BUDE docking successfully reproduces the linoleate complex and also supports binding of dexamethasone at the spike fatty acid site. Virtual screening of a library of approved drugs identifies vitamins D, K and A, as well as retinoid ligands with experimentally demonstrated activity against SARS-CoV-2 replication in vitro , as also potentially able to bind at this site. Our data suggest that the fatty acid binding site of the SARS-CoV-2 spike protein may bind a diverse array of candidate ligands. Targeting this site with small molecules, including dietary components such as vitamins, which may stabilise its locked conformation and represents a potential avenue for novel therapeutics or prophylaxis for COVID-19.

Unexpected free fatty acid binding pocket in the cryo-EM structure of SARS-CoV-2 spike protein (preprint)

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.