RESUMEN
Post-acute sequelae of COVID-19 (PASC, "Long COVID") pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.
Asunto(s)
Síndrome Post Agudo de COVID-19 , Serotonina , Humanos , COVID-19/complicaciones , Progresión de la Enfermedad , Inflamación , Síndrome Post Agudo de COVID-19/sangre , Síndrome Post Agudo de COVID-19/patología , Serotonina/sangre , VirosisRESUMEN
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection inhibits mitochondrial oxidative phosphorylation (OXPHOS) and elevates mitochondrial reactive oxygen species (ROS, mROS) which activates hypoxia-inducible factor-1alpha (HIF-1α), shifting metabolism toward glycolysis to drive viral biogenesis but also causing the release of mitochondrial DNA (mtDNA) and activation of innate immunity. To determine whether mitochondrially targeted antioxidants could mitigate these viral effects, we challenged mice expressing human angiotensin-converting enzyme 2 (ACE2) with SARS-CoV-2 and intervened using transgenic and pharmacological mitochondrially targeted catalytic antioxidants. Transgenic expression of mitochondrially targeted catalase (mCAT) or systemic treatment with EUK8 decreased weight loss, clinical severity, and circulating levels of mtDNA; as well as reduced lung levels of HIF-1α, viral proteins, and inflammatory cytokines. RNA-sequencing of infected lungs revealed that mCAT and Eukarion 8 (EUK8) up-regulated OXPHOS gene expression and down-regulated HIF-1α and its target genes as well as innate immune gene expression. These data demonstrate that SARS-CoV-2 pathology can be mitigated by catalytically reducing mROS, potentially providing a unique host-directed pharmacological therapy for COVID-19 which is not subject to viral mutational resistance.
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Antioxidantes , COVID-19 , Ratones Transgénicos , Mitocondrias , Fosforilación Oxidativa , SARS-CoV-2 , Animales , Ratones , COVID-19/virología , COVID-19/metabolismo , COVID-19/inmunología , COVID-19/patología , Antioxidantes/metabolismo , Antioxidantes/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Pulmón/virología , Pulmón/patología , Pulmón/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Catalasa/metabolismo , Catalasa/genética , Tratamiento Farmacológico de COVID-19 , Modelos Animales de Enfermedad , Inmunidad InnataRESUMEN
Rift Valley fever virus (RVFV) is an encephalitic bunyavirus that can infect neurons in the brain. There are no approved therapeutics that can protect from RVFV encephalitis. Innate immunity, the first line of defense against infection, canonically antagonizes viruses through interferon signaling. We found that interferons did not efficiently protect primary cortical neurons from RVFV, unlike other cell types. To identify alternative neuronal antiviral pathways, we screened innate immune ligands and discovered that the TLR2 ligand Pam3CSK4 inhibited RVFV infection, and other bunyaviruses. Mechanistically, we found that Pam3CSK4 blocks viral fusion, independent of TLR2. In a mouse model of RVFV encephalitis, Pam3CSK4 treatment protected animals from infection and mortality. Overall, Pam3CSK4 is a bunyavirus fusion inhibitor active in primary neurons and the brain, representing a new approach toward the development of treatments for encephalitic bunyavirus infections.
Asunto(s)
Lipopéptidos , Neuronas , Fiebre del Valle del Rift , Virus de la Fiebre del Valle del Rift , Animales , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Ratones , Lipopéptidos/farmacología , Fiebre del Valle del Rift/virología , Fiebre del Valle del Rift/prevención & control , Neuronas/metabolismo , Neuronas/virología , Ratones Endogámicos C57BL , Humanos , Inmunidad Innata/efectos de los fármacos , Encefalitis Viral/virología , Encefalitis Viral/inmunología , Encefalitis Viral/prevención & control , Encefalitis Viral/tratamiento farmacológico , Antivirales/farmacologíaRESUMEN
Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.
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COVID-19 , Ratones , Animales , Enzima Convertidora de Angiotensina 2/genética , SARS-CoV-2/metabolismo , Caquexia , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , HipoxiaRESUMEN
The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics.
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Adenosina Monofosfato , Adenosina , Alanina , Antivirales , COVID-19 , SARS-CoV-2 , Antivirales/farmacología , Antivirales/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/química , Humanos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Alanina/química , COVID-19/inmunología , COVID-19/virología , Animales , Agentes Inmunomoduladores/farmacología , Agentes Inmunomoduladores/química , Antagonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/química , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Pandemias , Tratamiento Farmacológico de COVID-19 , Chlorocebus aethiops , Replicación Viral/efectos de los fármacos , Células Vero , Betacoronavirus/efectos de los fármacos , Betacoronavirus/inmunología , Receptor de Adenosina A2A/metabolismo , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virologíaRESUMEN
Virulent pathogens often cause the release of host-derived damage-associated molecular patterns (DAMPs) from infected cells. During encounters with immune-evasive viruses that block inflammatory gene expression, preformed DAMPs provide backup inflammatory signals that ensure protective immunity. Whether DAMPs exhibit additional backup defense activities is unknown. Herein, we report that viral infection of barrier epithelia (keratinocytes) elicits the release of preformed interleukin-1 (IL-1) family cytokines, including the DAMP IL-1α. Mechanistic studies revealed that IL-1 acts on skin fibroblasts to induce an interferon (IFN)-like state that restricts viral replication. We identified a branch in the IL-1 signaling pathway that induces IFN-stimulated gene expression in infected cells and found that IL-1 signaling is necessary to restrict viral replication in human skin explants. These activities are most important to control immune-evasive virus replication in fibroblasts and other barrier cell types. These findings highlight IL-1 as an important backup antiviral system to ensure barrier defense.
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Evasión Inmune/inmunología , Interleucina-1/inmunología , Transducción de Señal/inmunología , Replicación Viral/inmunología , Animales , Línea Celular , Chlorocebus aethiops , Femenino , Fibroblastos/inmunología , Fibroblastos/virología , Expresión Génica/inmunología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Piel/inmunología , Piel/virología , Células VeroRESUMEN
Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.
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Células Epiteliales/inmunología , Células Epiteliales/virología , Inflamasomas/metabolismo , Intestinos/citología , Receptores Acoplados a Proteínas G/metabolismo , Infecciones por Rotavirus/inmunología , Infecciones por Rotavirus/virología , Rotavirus/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , ARN Helicasas DEAD-box/metabolismo , Células Epiteliales/metabolismo , Femenino , Inmunidad Innata , Inflamasomas/química , Inflamasomas/genética , Interleucina-18/inmunología , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión a Fosfato , Piroptosis , ARN Bicatenario/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/inmunología , Rotavirus/crecimiento & desarrolloRESUMEN
The central nervous system (CNS) necessitates intricately coordinated immune responses to prevent neurological disease. However, the emergence of viruses capable of entering the CNS and infecting neurons threatens this delicate balance. Our CNS is protected from foreign invaders and excess solutes by a semipermeable barrier of endothelial cells called the blood-brain barrier. Thereby, viruses have implemented several strategies to bypass this protective layer and modulate immune responses within the CNS. In this review, we outline these immune regulatory mechanisms and provide perspectives on future questions in this rapidly expanding field.
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Virus ARN , Virus , Barrera Hematoencefálica , Sistema Nervioso Central , Células Endoteliales , Inmunidad , ARNRESUMEN
The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle.IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein-protein interface in the capsid lattice. We also identify and characterize a mutation in the capsid protein that confers resistance to the inhibitor. This study reveals a novel mechanism by which a capsid-targeting small molecule can inhibit HIV-1 replication.
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Fármacos Anti-VIH/farmacología , Bencimidazoles/farmacología , Proteínas de la Cápside/metabolismo , Cápside/metabolismo , VIH-1/crecimiento & desarrollo , Ensamble de Virus/efectos de los fármacos , Sustitución de Aminoácidos/genética , Sitios de Unión/genética , Proteínas de la Cápside/genética , Línea Celular Tumoral , Proliferación Celular , Farmacorresistencia Viral/genética , Células HEK293 , VIH-1/genética , Humanos , Estructura Terciaria de Proteína , Transcripción Reversa/efectos de los fármacosRESUMEN
UNLABELLED: During virion maturation, HIV-1 capsid protein assembles into a conical core containing the viral ribonucleoprotein (vRNP) complex, thought to be composed mainly of the viral RNA and nucleocapsid protein (NC). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which is then incorporated into host chromosomes by integrase (IN) catalysis. Certain IN mutations (class II) and antiviral drugs (allosteric IN inhibitors [ALLINIs]) adversely affect maturation, resulting in virions that contain "eccentric condensates," electron-dense aggregates located outside seemingly empty capsids. Here we demonstrate that in addition to this mislocalization of electron density, a class II IN mutation and ALLINIs each increase the fraction of virions with malformed capsids (from â¼ 12% to â¼ 53%). Eccentric condensates have a high NC content, as demonstrated by "tomo-bubblegram" imaging, a novel labeling technique that exploits the susceptibility of NC to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. We conclude that eccentric condensates represent nonpackaged vRNPs and that either genetic or pharmacological inhibition of IN can impair vRNP incorporation into mature cores. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with internal electron density and the infectivity of a class II IN deletion mutant virus. Moreover, the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose a role for IN in initiating core morphogenesis and vRNP incorporation into the mature core during HIV-1 maturation. IMPORTANCE: Maturation, a process essential for HIV-1 infectivity, involves core assembly, whereby the viral ribonucleoprotein (vRNP, composed of vRNA and nucleocapsid protein [NC]) is packaged into a conical capsid. Allosteric integrase inhibitors (ALLINIs) affect multiple viral processes. We have characterized ALLINIs and integrase mutants that have the same phenotype. First, by comparing the effects of ALLINIs on several steps of the viral cycle, we show that inhibition of maturation accounts for compound potency. Second, by using cryoelectron tomography, we find that ALLINIs impair conical capsid assembly. Third, by developing tomo-bubblegram imaging, which specifically labels NC protein, we find that ALLINIs block vRNP packaging; instead, vRNPs form "eccentric condensates" outside the core. Fourth, malformed cores, typical of integrase-deleted virus, are partially replaced by conical cores when integrase is supplied in trans. Fifth, vRNA is necessary for ALLINI-induced eccentric condensate formation. These observations suggest that integrase is involved in capsid morphogenesis and vRNP packaging.
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Integrasa de VIH/metabolismo , VIH-1/fisiología , Proteínas de la Nucleocápside/metabolismo , Virión/fisiología , Ensamble de Virus/fisiología , Microscopía por Crioelectrón , Células HEK293 , VIH-1/metabolismo , Humanos , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa , Virión/metabolismoRESUMEN
Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inhibidores de Integrasa VIH , Integrasa de VIH/metabolismo , VIH-1/fisiología , Factores de Transcripción/metabolismo , Replicación Viral/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Regulación Alostérica/efectos de los fármacos , Línea Celular , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , Humanos , Factores de Transcripción/genética , Virión/genética , Virión/metabolismo , Replicación Viral/fisiologíaRESUMEN
BACKGROUND: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site. RESULTS: We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171. CONCLUSIONS: Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor.
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Acetatos/metabolismo , Farmacorresistencia Viral , Inhibidores de Integrasa VIH/metabolismo , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/enzimología , Mutación Missense , Quinolinas/metabolismo , Línea Celular , Cristalografía por Rayos X , Integrasa de VIH/química , Integrasa de VIH/genética , Histidina/genética , Histidina/metabolismo , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.
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Inhibidores de Integrasa VIH/farmacología , VIH-1/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Integración Viral , Acetatos/farmacología , Animales , Sitios de Unión , Línea Celular , Fibroblastos/metabolismo , Fibroblastos/virología , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Noqueados , Quinolinas/farmacologíaRESUMEN
Inappropriate type I interferon (IFN) signaling during embryo implantation and placentation is linked to poor pregnancy outcomes. Here, we evaluated the consequence of elevated type I IFN exposure on implantation using a biomimetic model of human implantation in an organ-on-a-chip device. We found that type I IFN reduced extravillous trophoblast (EVT) invasion capacity. Analyzing single-cell transcriptomes, we uncovered that IFN truncated endovascular EVT emergence in the implantation-on-a-chip device by stunting EVT epithelial-to-mesenchymal transition. Disruptions to the epithelial-to-mesenchymal transition is associated with the pathogenesis of preeclampsia, a life-threatening hypertensive disorder of pregnancy. Strikingly, unwarranted IFN stimulation induced genes associated with increased preeclampsia risk and a preeclamptic gene-like signature in EVTs. These dysregulated EVT phenotypes ultimately reduced EVT-mediated endothelial cell vascular remodeling in the implantation-on-a-chip device. Overall, our work indicates IFN signaling can alter EVT epithelial-to-mesenchymal transition progression which results in diminished EVT-mediated spiral artery remodeling and a preeclampsia gene signature upon sustained stimulation. Our work implicates unwarranted type I IFN as a maternal disturbance that can result in abnormal EVT function that could trigger preeclampsia.
RESUMEN
Red blood cells (RBCs) express the nucleic acid-binding toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-binding TLRs is unknown. Here we show that human RBCs express the RNA sensor TLR7. TLR7 is present on the red cell membrane and is associated with the RBC membrane protein Band 3. In patients with SARS-CoV2-associated sepsis, TLR7-Band 3 interactions in the RBC membrane are increased when compared with healthy controls. In vitro, RBCs bind synthetic ssRNA and RNA from ssRNA viruses. Thus, RBCs may serve as a previously unrecognized sink for exogenous RNA, expanding the repertoire of non-gas exchanging functions performed by RBCs.
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COVID-19 , Eritrocitos , SARS-CoV-2 , Receptor Toll-Like 7 , Humanos , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 7/genética , Eritrocitos/metabolismo , COVID-19/virología , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Sepsis/metabolismo , Sepsis/sangre , Sepsis/genética , Membrana Eritrocítica/metabolismo , Masculino , ARN/metabolismo , ARN/genética , FemeninoRESUMEN
Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. In the present study, we utilized a set of new mouse genetic tools developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID-19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells do not contribute significantly to the diverse vascular pathology associated with COVID-19.
RESUMEN
Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools 1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.
RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , COVID-19/inmunología , Núcleo Celular/inmunología , Factor 3 Regulador del Interferón/inmunología , Proteínas de Unión al ARN/inmunología , SARS-CoV-2/inmunología , Transducción de Señal/inmunología , Proteínas no Estructurales Virales/inmunología , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , COVID-19/genética , Núcleo Celular/genética , Células HeLa , Humanos , Factor 3 Regulador del Interferón/genética , FN-kappa B/genética , FN-kappa B/inmunología , Fosforilación/genética , Fosforilación/inmunología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas de Unión al ARN/genética , SARS-CoV-2/genética , Transducción de Señal/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2 fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2 fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2 fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortexâ" events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1 Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1 Cre ; LSL- hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.