ABSTRACT
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.
Subject(s)
Betacoronavirus/physiology , Heparitin Sulfate/metabolism , Peptidyl-Dipeptidase A/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Betacoronavirus/isolation & purification , Binding Sites , COVID-19 , Cell Line , Coronavirus Infections/pathology , Coronavirus Infections/virology , Heparin/chemistry , Heparin/metabolism , Heparitin Sulfate/chemistry , Humans , Kidney/metabolism , Lung/metabolism , Molecular Dynamics Simulation , Pandemics , Peptidyl-Dipeptidase A/chemistry , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Binding , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Virus InternalizationABSTRACT
The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.
Subject(s)
COVID-19 , Lung , Organoids , SARS-CoV-2 , Virus Internalization , Humans , SARS-CoV-2/physiology , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/virology , Lung/virology , Lung/immunology , Lung/pathology , Organoids/virology , COVID-19 Drug Treatment , Induced Pluripotent Stem Cells/virology , Angiotensin-Converting Enzyme 2/metabolism , Inflammation , Cytokines/metabolism , ApoptosisABSTRACT
BACKGROUND: Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. METHODS: We evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy. RESULTS: Genome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. CONCLUSIONS: Gain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.).
Subject(s)
Autoimmune Diseases , Dermatologic Agents , Janus Kinases , Scleroderma, Systemic , Janus Kinases/antagonists & inhibitors , Nitriles , Pyrazoles/therapeutic use , Pyrazoles/pharmacology , Pyrimidines , Scleroderma, Systemic/drug therapy , Scleroderma, Systemic/genetics , Autoimmune Diseases/drug therapy , Autoimmune Diseases/genetics , Mutation, Missense , Gain of Function Mutation , Dermatologic Agents/therapeutic use , Anti-Inflammatory Agents/therapeutic useABSTRACT
BACKGROUND: Hoffman syndrome is a syndromic, inborn error of immunity due to autosomal-dominant mutations in TOP2B, an essential gene required to alleviate topological stress during DNA replication and gene transcription. Although mutations identified in patients lead to a block in B-cell development and the absence of circulating B cells, an effect on natural killer (NK) cells was not previously examined. OBJECTIVE: We sought to determine whether disease-associated mutations in TOP2B impact NK-cell development and function. METHODS: Using a knockin murine model and patient-derived induced pluripotent stem cells (iPSCs), we investigated NK-cell development in mouse bone marrow and spleen, and performed immunophenotyping by flow cytometry, gene expression, and functional assessment of cytotoxic activity in murine NK cells, and human IPSC-derived NK cells. RESULTS: Mature NK cells were reduced in the periphery of TOP2B knockin mice consistent with patient reports, with reduced cytotoxicity toward target cell lines. IPSCs were successfully derived from patients with Hoffman syndrome, but under optimal conditions showed reduced cytotoxicity compared with iPSC-derived NK cells from healthy controls. CONCLUSIONS: Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function in murine and human models.
Subject(s)
Induced Pluripotent Stem Cells , Killer Cells, Natural , Animals , Cell Line , Craniofacial Abnormalities , Humans , Induced Pluripotent Stem Cells/metabolism , Limb Deformities, Congenital , Mice , Mutation , Primary Immunodeficiency Diseases , Urogenital AbnormalitiesABSTRACT
Remdesivir (RDV; GS-5734) is currently the only FDA-approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The drug is approved for use in adults or children 12 years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2-infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed, and several, including molnupiravir and PF-07321332, are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of remdesivir nucleoside (RVn; GS-441524) that are processed to RVn monophosphate, the precursor of the active RVn triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma, and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types, including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells, and Huh7.5 cells. In Syrian hamsters, oral treatment with 1-O-octadecyl-2-O-benzyl-glycero-3-phosphate RVn (ODBG-P-RVn) was well tolerated and achieved therapeutic levels in plasma above the 90% effective concentration (EC90) for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.
Subject(s)
COVID-19 Drug Treatment , Prodrugs , Adenosine/analogs & derivatives , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Antiviral Agents/pharmacology , Caco-2 Cells , Cricetinae , Humans , Lipids , SARS-CoV-2ABSTRACT
Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn-2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections.
Subject(s)
Antiviral Agents , COVID-19 , Animals , Mice , SARS-CoV-2 , PhospholipidsABSTRACT
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.
ABSTRACT
The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.
ABSTRACT
The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/ß. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.
ABSTRACT
Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFRα). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.
Subject(s)
Alveolar Epithelial Cells/cytology , Endoderm/cytology , Induced Pluripotent Stem Cells/cytology , Lung/cytology , Organogenesis , Organoids/cytology , Tissue Engineering/methods , Alveolar Epithelial Cells/metabolism , Cell Culture Techniques , Cell Differentiation , Developmental Biology , Endoderm/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Lung/metabolism , Organoids/metabolismABSTRACT
Remdesivir (RDV, GS-5734) is currently the only FDA-approved antiviral drug for the treatment of SARS CoV-2 infection. The drug is approved for use in adults or children 12-years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2 infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed and several including molnupiravir and PF-07321332 are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of Remdesivir nucleoside (RVn, GS-441524) that are processed to RVn-monophosphate, the precursor of the active RVn-triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells and Huh7.5 cells. In Syrian hamsters oral treatment with ODBG-P-RVn was well tolerated and achieved therapeutic levels in plasma above the EC90 for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.
ABSTRACT
We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.
ABSTRACT
The normal development of the pulmonary system is critical to transitioning from placental-dependent fetal life to alveolar-dependent newborn life. Human lung development and disease have been difficult to study due to the lack of an in vitro model system containing cells from the large airways and distal alveolus. This article describes a system that allows human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to differentiate and form three-dimensional (3D) structures that emulate the development, cytoarchitecture, and function of the lung ("organoids"), containing epithelial and mesenchymal cell populations, and including the production of surfactant and presence of ciliated cells. The organoids can also be invested with mesoderm derivatives, differentiated from the same human pluripotent stem cells, such as alveolar macrophages and vasculature. Such lung organoids may be used to study the impact of environmental modifiers and perturbagens (toxins, microbial or viral pathogens, alterations in microbiome) or the efficacy and safety of drugs, biologics, and gene transfer. © 2020 Wiley Periodicals LLC. Basic Protocol: hESC/hiPSC dissection, definitive endoderm formation, and lung progenitor cell induction.
Subject(s)
Coronavirus Infections/pathology , Lung/cytology , Organoids/cytology , Pneumonia, Viral/pathology , Respiratory Tract Infections/pathology , Betacoronavirus , COVID-19 , Cell Culture Techniques , Cell Differentiation , Coronavirus Infections/therapy , Endoderm/cytology , Human Embryonic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Lung/growth & development , Lung/physiology , Models, Biological , Pandemics , Patient-Specific Modeling , Pneumonia, Viral/therapy , Respiratory Tract Infections/therapy , SARS-CoV-2 , Time-Lapse ImagingABSTRACT
One approach to understanding how tissue-specific cancers emerge is to determine the requirements for "reprograming" such neoplastic cells back to their developmentally normal primordial pre-malignant epiblast-like pluripotent state and then scrutinizing their spontaneous reconversion to a neoplasm, perhaps rendering salient the earliest pivotal oncogenic pathway(s) (before other aberrations accumulate in the adult tumor). For the prototypical malignancy anaplastic thyroid carcinoma (ATC), we found that tonic RAS reduction was obligatory for reprogramming cancer cells to a normal epiblast-emulating cells, confirmed by changes in their transcriptomic and epigenetic profiles, loss of neoplastic behavior, and ability to derive normal somatic cells from their "epiblast organoids." Without such suppression, ATCs re-emerged from the clones. Hence, for ATC, RAS inhibition was its "reprogram enablement" (RE) factor. Each cancer likely has its own RE factor; identifying it may illuminate pre-malignant risk markers, better classifications, therapeutic targets, and tissue-specification of a previously pluripotent, now neoplastic, cell.