We show that SARS-CoV-2 spike protein interacts with both cellular heparan sulfate and angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD). Docking studies suggest a ...heparin/heparan sulfate-binding site adjacent to the ACE2-binding site. Both ACE2 and heparin can bind independently to spike protein in vitro, and a ternary complex can be generated using heparin as a scaffold. Electron micrographs of spike protein suggests that heparin enhances the open conformation of the RBD that binds ACE2. On cells, spike protein binding depends on both heparan sulfate and ACE2. Unfractionated heparin, non-anticoagulant heparin, heparin lyases, and lung heparan sulfate potently block spike protein binding and/or infection by pseudotyped virus and authentic SARS-CoV-2 virus. We suggest a model in which viral attachment and infection involves heparan sulfate-dependent enhancement of binding to ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin presents new therapeutic opportunities.
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•SARS-CoV-2 spike protein interacts with heparan sulfate and ACE2 through the RBD•Heparan sulfate promotes Spike-ACE2 interaction•SARS-CoV-2 infection is co-dependent on heparan sulfate and ACE2•Heparin and non-anticoagulant derivatives block SARS-CoV-2 binding and infection
Clausen et al. provide evidence that heparan sulfate is a necessary co-factor for SARS-CoV-2 infection. They show that heparan sulfate interacts with the receptor-binding domain of the SARS-CoV-2 spike glycoprotein, adjacent to ACE2, shifting the spike structure to an open conformation to facilitate ACE2 binding.
Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using ...HLA class I and II predicted peptide “megapools,” circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%–27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in ∼40%–60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating “common cold” coronaviruses and SARS-CoV-2.
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•Measuring immunity to SARS-CoV-2 is key for understanding COVID-19 and vaccine development•Epitope pools detect CD4+ and CD8+ T cells in 100% and 70% of convalescent COVID patients•T cell responses are focused not only on spike but also on M, N, and other ORFs•T cell reactivity to SARS-CoV-2 epitopes is also detected in non-exposed individuals
An analysis of immune cell responses to SARS-CoV-2 from recovered patients identifies the regions of the virus that is targeted and also reveals cross-reactivity with other common circulating coronaviruses
Clinical evidence suggests the central nervous system is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although the mechanisms are unclear. Pericytes are perivascular ...cells within the brain that are proposed as SARS-CoV-2 infection points. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Before infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model that supports astrocytic maturation as well as SARS-CoV-2 entry and replication in neural tissue; thus, PCCOs serve as an experimental model for neural infection.
The emergence of Zika virus (ZIKV) as a global health threat has highlighted the unmet need for ZIKV-specific vaccines and antiviral treatments. ZIKV infects dendritic cells (DC), which have pivotal ...functions in activating innate and adaptive antiviral responses; however, the mechanisms by which DC function is subverted to establish ZIKV infection are unclear. Here we develop a genomics profiling method that enables discrete analysis of ZIKV-infected versus neighboring, uninfected primary human DCs to increase the sensitivity and specificity with which ZIKV-modulated pathways can be identified. The results show that ZIKV infection specifically increases the expression of genes enriched for lipid metabolism-related functions. ZIKV infection also increases the recruitment of sterol regulatory element-binding protein (SREBP) transcription factors to lipid gene promoters, while pharmacologic inhibition or genetic silencing of SREBP2 suppresses ZIKV infection of DCs. Our data thus identify SREBP2-activated transcription as a mechanism for promoting ZIKV infection amenable to therapeutic targeting.
Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly ...performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.
Coronavirus disease 2019 (COVID‐19) is caused by SARS‐CoV‐2 and has spread across the globe. SARS‐CoV‐2 is a highly infectious virus with no vaccine or antiviral therapy available to control the ...pandemic; therefore, it is crucial to understand the mechanisms of viral pathogenesis and the host immune responses to SARS‐CoV‐2. SARS‐CoV‐2 is a new member of the betacoronavirus genus like other closely related viruses including SARS‐CoV and Middle East respiratory syndrome coronavirus (MERS‐CoV). Both SARS‐CoV and MERS‐CoV have caused serious outbreaks and epidemics in the past eighteen years. Here, we report that one of the interferon‐stimulated genes (ISGs), cholesterol 25‐hydroxylase (CH25H), is induced by SARS‐CoV‐2 infection in vitro and in COVID‐19‐infected patients. CH25H converts cholesterol to 25‐hydrocholesterol (25HC) and 25HC shows broad anti‐coronavirus activity by blocking membrane fusion. Furthermore, 25HC inhibits USA‐WA1/2020 SARS‐CoV‐2 infection in lung epithelial cells and viral entry in human lung organoids. Mechanistically, 25HC inhibits viral membrane fusion by activating the ER‐localized acyl‐CoA:cholesterol acyltransferase (ACAT) which leads to the depletion of accessible cholesterol from the plasma membrane. Altogether, our results shed light on a potentially broad antiviral mechanism by 25HC through depleting accessible cholesterol on the plasma membrane to suppress virus–cell fusion. Since 25HC is a natural product with no known toxicity at effective concentrations, it provides a potential therapeutic candidate for COVID‐19 and emerging viral diseases in the future.
Synopsis
25‐Hydroxycholesterol, produced by the interferon‐stimulated protein CH25H, inhibits the entry of SARS‐CoV‐2, SARS‐CoV, and MERS‐CoV. Mechanistically, 25HC activates the ER‐localized enzyme ACAT, whose activity depletes accessible cholesterol on the plasma membrane and restricts viral membrane fusion.
An interferon‐stimulated gene, CH25H is induced in SARS‐CoV-2 infection in vitro and in vivo.
25HC exhibits broad anti‐coronavirus activity by blocking membrane fusion.
25HC activates ACAT to induce internalization of accessible cholesterol on the plasma membrane and restricts viral fusion.
25HC inhibits USA‐WA1/2020 SARS‐CoV-2 infection in lung epithelial cells and virus entry in human lung organoids.
Interferon‐induced cholesterol hydroxylation, and subsequent activation of the ER‐localized ACAT enzyme that depletes accessible cholesterol, represents a new host defence mechanism restricting membrane fusion and entry of severely pathogenic coronaviruses.
We isolated a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2 variant from a person with coronavirus disease 2019 recrudescence after nirmatrelvir/ritonavir treatment. Antiviral ...sensitivity and neutralizing antibody testing were performed with both parental SARS-CoV-2 and multiple variants of concern. We found that neither nirmatrelvir resistance nor absence of neutralizing immunity was a likely cause of the recrudescence.
COVID-19 is a transmissible respiratory disease caused by a novel coronavirus, SARS-CoV-2, and has become a global health emergency. There is an urgent need for robust and practical in vitro model ...systems to investigate viral pathogenesis. Here, we generated human induced pluripotent stem cell (iPSC)-derived lung organoids (LORGs), cerebral organoids (CORGs), neural progenitor cells (NPCs), neurons, and astrocytes. LORGs containing epithelial cells, alveolar types 1 and 2, highly express ACE2 and TMPRSS2 and are permissive to SARS-CoV-2 infection. SARS-CoV-2 infection induces interferons, cytokines, and chemokines and activates critical inflammasome pathway genes. Spike protein inhibitor, EK1 peptide, and TMPRSS2 inhibitors (camostat/nafamostat) block viral entry in LORGs. Conversely, CORGs, NPCs, astrocytes, and neurons express low levels of ACE2 and TMPRSS2 and correspondingly are not highly permissive to SARS-CoV-2 infection. Infection in neuronal cells activates TLR3/7, OAS2, complement system, and apoptotic genes. These findings will aid in understanding COVID-19 pathogenesis and facilitate drug discovery.
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•Lung organoids express ACE2/TMPRSS2 and are permissive to SARS-CoV-2 infection•SARS-CoV-2 induces IFNs and cytokines/chemokines, and activates inflammasome pathway•SARS-CoV-2 infection in neurons activates TLR3/7, OAS2, and apoptotic genes•Spike protein and TMPRSS2 inhibitors reduce SARS-CoV-2 infection in organoids
In this report, Rana and colleagues show that human iPSC-derived lung and brain organoids express ACE2/TMPRSS2 and are permissive to SARS-CoV-2 infection inducing specific pathways in lung and brain cells. Spike protein inhibitor, EK1 peptide, and TMPRSS2 inhibitors (camostat/nafamostat) reduced SARS-CoV-2 infection in lung organoids, providing a model system for drug development.
Macrophages play pivotal roles in both the induction and resolution phases of inflammatory processes. Macrophages have been shown to synthesize anti-inflammatory fatty acids in an LXR-dependent ...manner, but whether the production of these species contributes to the resolution phase of inflammatory responses has not been established. Here, we identify a biphasic program of gene expression that drives production of anti-inflammatory fatty acids 12–24 hr following TLR4 activation and contributes to downregulation of mRNAs encoding pro-inflammatory mediators. Unexpectedly, rather than requiring LXRs, this late program of anti-inflammatory fatty acid biosynthesis is dependent on SREBP1 and results in the uncoupling of NFκB binding from gene activation. In contrast to previously identified roles of SREBP1 in promoting production of IL1β during the induction phase of inflammation, these studies provide evidence that SREBP1 also contributes to the resolution phase of TLR4-induced gene activation by reprogramming macrophage lipid metabolism.
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•TLR ligands induce reciprocal inflammatory and lipid biosynthetic gene expression•SREBP1 levels and activity are increased 12–24 hr following TLR4 activation•SREBP1 drives the production of anti-inflammatory fatty acids in macrophages•Loss of SREBP1 results in impaired resolution of inflammatory responses
Oishi and Spann et al. identify a biphasic macrophage gene expression program underlying anti-inflammatory fatty acid production following TLR4 activation. The late anti-inflammatory program is dependent on SREBP1. This is surprising given the known roles of SREBP1 in promoting IL1β production during the induction of inflammation and highlights its dual roles.
The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild ...versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.
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