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The interactions between severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and human host factors enable the virus to propagate infections that lead to COVID-19. The spike protein is the largest structural component of the virus and mediates interactions essential for infection, including with the primary ACE2 receptor. We performed two independent cell-based systematic screens to determine whether there are additional proteins by which the spike protein of SARS-CoV-2 can interact with human cells. We discovered that in addition to ACE2, expression of LRRC15 also causes spike protein binding. This interaction is distinct from other known spike attachment mechanisms such as heparan sulfates or lectin receptors. Measurements of orthologous coronavirus spike proteins implied the interaction was restricted to SARS-CoV-2, suggesting LRRC15 represents a novel class of spike binding interaction. We localized the interaction to the C-terminus of the S1 domain, and showed that LRRC15 shares recognition of the ACE2 receptor binding domain. From analyzing proteomics and single-cell transcriptomics, we identify LRRC15 expression as being common in human lung vasculature cells and fibroblasts. Although infection assays demonstrated that LRRC15 alone is not sufficient to permit viral entry, we present evidence it can modulate infection of human cells. This unexpected interaction merits further investigation to determine how SARS-CoV-2 exploits host LRRC15 and whether it could account for any of the distinctive features of COVID-19. In briefWe present evidence from genome-wide screening that the spike protein of SARS-CoV-2 interacts with human cells expressing LRRC15. The interaction is distinct from previously known classes of spike attachment factors, and appears to have emerged recently within the coronavirus family. Although not sufficient for cell invasion, this interaction can modulate viral infection. Our data point to an unappreciated host factor for SARS-CoV-2, with potential relevance to COVID-19. Highlights- Two systematic cell-based screens for SARS-CoV-2 spike protein binding identify LRRC15 as a human host factor - Interaction with LRRC15 is reproducible in different human cell lines and independent of known glycan or ACE2 binding pathways - The C-terminal S1 domain of SARS-CoV-2 spike binds LRRC15 with sub-micromolar affinity, while related coronavirus spikes do not - LRRC15 is expressed in tissues with high ACE2 levels and may modulate infection
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Cigarette smoking has multiple serious negative health consequences. However, the epidemiological relationship between cigarette smoking and SARS-CoV-2 infection is controversial; and the interaction between cigarette smoking, airway expression of the ACE2 receptor and the susceptibility of airway cells to infection is unclear. We exposed differentiated air-liquid interface cultures derived from primary human airway stem cells to cigarette smoke extract (CSE) and infected them with SARS-CoV-2. We found that CSE increased expression of full-length ACE2 (flACE2) but did not alter the expression of a Type I-interferon sensitive truncated ACE2 that lacks the capacity to bind SARS-CoV-2 or a panel of interferon-sensitive genes. Importantly, exposure to CSE did not increase viral infectivity despite the increase in flACE2. Our data are consistent with epidemiological data suggesting current smokers are not at excess risk of SARS-CoV-2 infection. This does not detract from public health messaging emphasising the excess risk of severe COVID-19 associated with smoking-related cardiopulmonary disease.
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Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This brought to mind the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. We demonstrated the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes we also observed in B cell VHL-deficient mice. This was corroborated by hypoxia-related transcriptional changes in COVID-19 patients, and by similar B cell abnormalities in mice kept in hypoxic conditions, including reduced marginal zone and germinal center B cells. Thus hypoxia might contribute to B cell pathology in COVID-19, and in other hypoxic states. Through this mechanism it may impact on COVID-19 outcome, and be remediable through early oxygen therapy.
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BackgroundThere are limited effective prophylactic treatments for SARS-CoV-2 infection, and limited early treatment options. Viral cell entry requires spike protein binding to the ACE2 receptor and spike cleavage by TMPRSS2, a cell surface serine protease. Targeting of TMPRSS2 by either androgen blockade or direct inhibition is already in clinical trials in early SARS-CoV-2 infection. MethodsThe likely initial cells of SARS-CoV-2 entry are the ciliated cells of the upper airway. We therefore used differentiated primary human airway epithelial cells maintained at the air-liquid interface (ALI) to test the impact of targeting TMPRSS2 on the prevention of SARS-CoV-2 infection. ResultsWe first modelled the systemic delivery of compounds. Enzalutamide, an oral androgen receptor antagonist, had no impact on SARS-Cov-2 infection. By contrast, camostat mesylate, an orally available serine protease inhibitor, blocked SARS-CoV-2 entry. However, camostat is rapidly metabolised in the circulation in vivo, and systemic bioavailability after oral dosing is low. We therefore modelled local airway administration by applying camostat to the apical surface of the differentiated ALI cultures. We demonstrated that a brief exposure to topical camostat is effective at restricting SARS-CoV-2 viral infection. ConclusionThese experiments demonstrate a potential therapeutic role for topical camostat for pre- or post-exposure prophylaxis of SARS-CoV-2, which can now be evaluated in a clinical trial.
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Severe Covid-19 is associated with elevated plasma Factor V (FV) and increased risk of thromboembolism. We report that neutrophils, T regulatory cells (Tregs), and monocytes from patients with severe Covid-19 express FV, and expression correlates with T cell lymphopenia. In vitro full length FV, but not FV activated by thrombin cleavage, suppresses T cell proliferation. Increased and prolonged FV expression by cells of the innate and adaptive immune systems may contribute to lymphopenia in severe Covid-19. Activation by thrombin destroys the immunosuppressive properties of FV. Anticoagulation in Covid-19 patients may have the unintended consequence of suppressing the adaptive immune system.
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The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. S exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor binding site, and subsequently from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here we have designed mutations in S which allow production of thermostable, crosslinked, S protein trimers that are trapped in the closed, pre-fusion, state. We have determined the structures of crosslinked and non-crosslinked proteins, identifying two distinct closed conformations of the S trimer. We demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.
