RESUMEN
Most enveloped viruses infect cells by binding receptors at the cell surface and undergo trafficking through the endocytic pathway to a compartment with the requisite conditions to trigger fusion with a host endosomal membrane. Broad categories of compartments in the endocytic pathway include early and late endosomes, which can be further categorized into subpopulations with differing rates of maturation and motility characteristics. Endocytic compartments have varying protein and lipid components, luminal ionic conditions and pH that provide uniquely hospitable environments for specific viruses to fuse. In order to characterize compartments that permit fusion, we studied the trafficking and fusion of viral particles pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) on their surface and equipped with a novel pH sensor and a fluorescent content marker to measure pH, motion and fusion at the single particle level in live cells. We found that the VSV-G particles fuse predominantly from more acidic and more motile endosomes, and that a significant fraction of particles is trafficked to more static and less acidic endosomes that do not support their fusion. Moreover, the fusion-supporting endosomes undergo directed motion.
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Estomatitis Vesicular , Internalización del Virus , Animales , Endocitosis , Endosomas/metabolismo , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Estomatitis Vesicular/metabolismoRESUMEN
Existing pharmacodynamic (PD) mathematical models for drug combinations discriminate antagonistic, additive, multiplicative, and synergistic effects, but fail to consider how concentration-dependent drug interaction effects may vary across an entire dose-response matrix. We developed a two-way pharmacodynamic (TWPD) model to capture the PD of two-drug combinations. TWPD captures interactions between upstream and downstream drugs that act on different stages of viral replication, by quantifying upstream drug efficacy and concentration-dependent effects on downstream drug pharmacodynamic parameters. We applied TWPD to previously published in vitro drug matrixes for repurposed potential anti-Ebola and anti-SARS-CoV-2 drug pairs. Depending on the drug pairing, the model recapitulated combined efficacies as or more accurately than existing models and can be used to infer efficacy at untested drug concentrations. TWPD fits the data slightly better in one direction for all drug pairs, meaning that we can tentatively infer the upstream drug. Based on its high accuracy, TWPD could be used in concert with PK models to estimate the therapeutic effects of drug pairs in vivo.
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COVID-19 , Fiebre Hemorrágica Ebola , Humanos , Modelos Biológicos , SARS-CoV-2 , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Combinación de MedicamentosRESUMEN
The ongoing coronavirus disease 2019 (COVID-19) pandemic has highlighted the need to better understand virus-host interactions. We developed a network-based method that expands the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-host protein interaction network and identifies host targets that modulate viral infection. To disrupt the SARS-CoV-2 interactome, we systematically probed for potent compounds that selectively target the identified host proteins with high expression in cells relevant to COVID-19. We experimentally tested seven chemical inhibitors of the identified host proteins for modulation of SARS-CoV-2 infection in human cells that express ACE2 and TMPRSS2. Inhibition of the epigenetic regulators bromodomain-containing protein 4 (BRD4) and histone deacetylase 2 (HDAC2), along with ubiquitin-specific peptidase (USP10), enhanced SARS-CoV-2 infection. Such proviral effect was observed upon treatment with compounds JQ1, vorinostat, romidepsin and spautin-1, when measured by cytopathic effect and validated by viral RNA assays, suggesting that the host proteins HDAC2, BRD4 and USP10 have antiviral functions. We observed marked differences in antiviral effects across cell lines, which may have consequences for identification of selective modulators of viral infection or potential antiviral therapeutics. While network-based approaches enable systematic identification of host targets and selective compounds that may modulate the SARS-CoV-2 interactome, further developments are warranted to increase their accuracy and cell-context specificity.
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Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Humanos , Mapas de Interacción de Proteínas , Proteínas Nucleares , Factores de Transcripción , Antivirales/farmacología , Ubiquitina Tiolesterasa , Proteínas de Ciclo CelularRESUMEN
BACKGROUND: The aim of the study was to use bioelectrical impedance analysis (BIA) to assess nutritional status of elderly patients admitted to hospital and quantitatively measure the impact of the Cardiff and Vale University Health Board (CAVUHB) model ward. Secondary objectives were to assess the feasibility of using BIA in this patient population and compare nutrition risk screening tools against fat-free mass index (FFMI). METHODS: A prospective, comparative, single-centre, service evaluation of a 'model ward for nutrition and hydration' undertaken in medical and rehabilitation beds in a large UK teaching hospital. RESULTS: A total of 450 BIA measurements were taken using a Bodystat Multiscan 5000 on 162 patients; several patients had repeated measurements during their hospital stay. Patients tolerated the procedure well, but lack of accurate weight, implanted medical devices and tissue viability precluded some participants. CONCLUSIONS: BIA is quick, non-invasive, simple to complete and can elicit huge data about an individual's body composition. In a larger cohort of medical admissions, BIA could assist in identifying the sensitivity and specificity of the nutrition screening tools. The collective benefit of a series of nutritional interventions preserved nutritional status better in this elderly inpatient population than usual models of care. Although results were not statistically significant, there is an opportunity with the new model of care to better support frail patients and prevent deconditioning.
RESUMEN
To enter a cell and establish infection, HIV must first fuse its lipid envelope with the host cell plasma membrane. Whereas the process of HIV membrane fusion can be tracked by fluorescence microscopy, the 3D configuration of proteins and lipids at intermediate steps can only be resolved with cryo-electron tomography (cryoET). However, cryoET of whole cells is technically difficult. To overcome this problem, we have adapted giant plasma membrane vesicles (or blebs) from native cell membranes expressing appropriate receptors as targets for fusion with HIV envelope glycoprotein-expressing pseudovirus particles with and without Serinc host restriction factors. The fusion behavior of these particles was probed by TIRF microscopy on bleb-derived supported membranes. Timed snapshots of fusion of the same particles with blebs were examined by cryo-ET. The combination of these methods allowed us to characterize the structures of various intermediates on the fusion pathway and showed that when Serinc3 or Serinc5 (but not Serinc2) were present, later fusion products were more prevalent, suggesting that Serinc3/5 act at multiple steps to prevent progression to full fusion. In addition, the antifungal amphotericin B reversed Serinc restriction, presumably by intercalation into the fusing membranes. Our results provide a highly detailed view of Serinc restriction of HIV-cell membrane fusion and thus extend current structural and functional information on Serinc as a lipid-binding protein.
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Membrana Celular/metabolismo , Microscopía por Crioelectrón , VIH-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Línea Celular , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Fusión de Membrana , Microscopía Fluorescente , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus [JUNV], lymphocytic choriomeningitis virus [LCMV], and Pichinde virus [PICV]). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks.
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Antivirales/uso terapéutico , Infecciones por Arenaviridae/tratamiento farmacológico , Arenavirus/efectos de los fármacos , Administración Oral , Animales , Infecciones por Arenaviridae/virología , Línea Celular , Chlorocebus aethiops , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Células HEK293 , Humanos , Ratones , Prueba de Estudio Conceptual , Células VeroRESUMEN
AIM: The use of standard CO2 for insufflation during laparoscopic colorectal surgery may be associated with cooling and drying of the peritoneal cavity, contributing to perioperative hypothermia. The aim of this work was the assess the feasibility of a study to compare insufflation of warmed, humidified CO2 (WHCO2) (using HumiGard, Fisher and Paykel Healthcare) with standard measures and its impact on the quality of recovery of surgical patients. METHOD: A single-centre, triple-blind, feasibility, randomized controlled trial (RCT) of adults scheduled for planned laparoscopic colorectal surgery. The primary outcome was recruitment. Secondary outcomes included feasibility of blinding, acceptability to patients and suitability of objective measures: patient-reported quality of recovery using a validated questionnaire (QoR-40), patient pain scores and semi-continuous core temperature measurements. RESULTS: Thirty-nine participants were randomized to either the WHCO2 group (n = 19) or standard care alone (n = 20). Recruitment to the study was successful and acceptable to patients. Blinding of the surgeons, patients and assessors was effective. Response rates to QoR-40 were high but ceiling effects were observed, indicating that the tool was unsuitable in this population. Fewer patients in the WHCO2 group reported postoperative nausea and vomiting (PONV) at days 1 (53% vs. 65%) and 3 (37% vs. 60%). The median hospital length of stay was 5.5 days in the standard care group and 4 days in the WHCO2 group. CONCLUSION: A study of WHCO2 for insufflation in laparoscopic colorectal surgery would be highly acceptable to both patients and researchers. Potential reductions in PONV and hospital length of stay in patients treated with WHCO2 merit further investigation. The design of the full-scale RCT will benefit from this feasibility study.
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Neoplasias Colorrectales , Hipotermia , Insuflación , Laparoscopía , Adulto , Dióxido de Carbono , Estudios de Factibilidad , Humanos , Humedad , Hipotermia/etiología , Hipotermia/prevención & control , Dolor Postoperatorio/etiología , Dolor Postoperatorio/prevención & controlRESUMEN
The cardiac muscle ryanodine receptor-Ca2+ release channel (RyR2) constitutes the sarcoplasmic reticulum (SR) Ca2+ efflux mechanism that initiates myocyte contraction, while cardiac myosin-binding protein-C (cMyBP-C; also known as MYBPC3) mediates regulation of acto-myosin cross-bridge cycling. In this paper, we provide the first evidence for the presence of direct interaction between these two proteins, forming a RyR2-cMyBP-C complex. The C-terminus of cMyBP-C binds with the RyR2 N-terminus in mammalian cells and the interaction is not mediated by a fibronectin-like domain. Notably, we detected complex formation between both recombinant cMyBP-C and RyR2, as well as between the native proteins in cardiac tissue. Cellular Ca2+ dynamics in HEK293 cells is altered upon co-expression of cMyBP-C and RyR2, with lowered frequency of RyR2-mediated spontaneous Ca2+ oscillations, suggesting that cMyBP-C exerts a potential inhibitory effect on RyR2-dependent Ca2+ release. Discovery of a functional RyR2 association with cMyBP-C provides direct evidence for a putative mechanistic link between cytosolic soluble cMyBP-C and SR-mediated Ca2+ release, via RyR2. Importantly, this interaction may have clinical relevance to the observed cMyBP-C and RyR2 dysfunction in cardiac pathologies, such as hypertrophic cardiomyopathy.
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Proteínas Portadoras/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Calcio/metabolismo , Señalización del Calcio/fisiología , Citosol/metabolismo , Células HEK293 , Humanos , Unión Proteica , Retículo Sarcoplasmático/metabolismoRESUMEN
Influenza virus is an RNA virus encapsulated in a lipid bilayer derived from the host cell plasma membrane. Previous studies showed that influenza virus infection depends on cellular lipids, including the sphingolipids sphingomyelin and sphingosine. Here we examined the role of a third sphingolipid, glucosylceramide, in influenza virus infection following clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR-Cas9)-mediated knockout (KO) of its metabolizing enzyme glucosylceramidase (GBA). After confirming GBA knockout of HEK 293 and A549 cells by both Western blotting and lipid mass spectrometry, we observed diminished infection in both KO cell lines by a PR8 (H1N1) green fluorescent protein (GFP) reporter virus. We further showed that the reduction in infection correlated with impaired influenza virus trafficking to late endosomes and hence with fusion and entry. To examine whether GBA is required for other enveloped viruses, we compared the results seen with entry mediated by the glycoproteins of Ebola virus, influenza virus, vesicular stomatitis virus (VSV), and measles virus in GBA knockout cells. Entry inhibition was relatively robust for Ebola virus and influenza virus, modest for VSV, and mild for measles virus, suggesting a greater role for viruses that enter cells by fusing with late endosomes. As the virus studies suggested a general role for GBA along the endocytic pathway, we tested that hypothesis and found that trafficking of epidermal growth factor (EGF) to late endosomes and degradation of its receptor were impaired in GBA knockout cells. Collectively, our findings suggest that GBA is critically important for endocytic trafficking of viruses as well as of cellular cargos, including growth factor receptors. Modulation of glucosylceramide levels may therefore represent a novel accompaniment to strategies to antagonize "late-penetrating" viruses, including influenza virus.IMPORTANCE Influenza virus is the pathogen responsible for the second largest pandemic in human history. A better understanding of how influenza virus enters host cells may lead to the development of more-efficacious therapies against emerging strains of the virus. Here we show that the glycosphingolipid metabolizing enzyme glucosylceramidase is required for optimal influenza virus trafficking to late endosomes and for consequent fusion, entry, and infection. We also provide evidence that promotion of influenza virus entry by glucosylceramidase extends to other endosome-entering viruses and is due to a general requirement for this enzyme, and hence for optimal levels of glucosylceramide, for efficient trafficking of endogenous cargos, such as the epidermal growth factor (EGF) receptor, along the endocytic pathway. This work therefore has implications for the basic process of endocytosis as well as for pathogenic processes, including virus entry and Gaucher disease.
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Endocitosis/fisiología , Glucosilceramidasa/metabolismo , Orthomyxoviridae/metabolismo , Células A549 , Ebolavirus/metabolismo , Endosomas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Glucosilceramidasa/fisiología , Células HEK293 , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Virus de la Influenza A/fisiología , Virus del Sarampión/metabolismo , Internalización del VirusRESUMEN
BACKGROUND: Previously, routine antenatal anti-D prophylaxis (RAADP) was administered to all RhD-negative mothers to reduce the risk of sensitisation in the UK's National Health Service (NHS). If the baby is RhD-negative, RAADP is not required. In 2016, the UK National Institute for Health and Care Excellence (NICE) recommended non-invasive prenatal testing (NIPT) for fetal RHD genotype as a cost-effective option to guide RAADP. OBJECTIVES: To evaluate the implementation of high-throughput NIPT for fetal RHD genotype in maternity units in England by addressing research recommendations from the NICE. These were to reduce uncertainty around the resource use and cost of staff training, management of samples and results and record-keeping, as well as resultant changes to antenatal or post-partum care and performance of NIPT. METHODS: A cross-sectional survey was developed and sent to clinicians at 39 English NHS Trusts in May 2018. Qualitative interviews with seven individuals were conducted to explore missing or contraindicatory data. Qualitative findings were supplemented with NIPT test results (April 2017 to February 2019) from English hospitals. RESULTS: Staff reported that training took up to 30 minutes. There were no extra costs associated with sample management or additional appointments. Extra time required for record-keeping and management of test results was balanced later in the patient pathway. The antenatal pathway was not changed in the Trusts surveyed. The survey revealed that four post-partum scenarios were being used within English NHS Trusts. The frequency of inconclusive NIPT results was 4.3%. CONCLUSION: NIPT for fetal RHD genotype can be implemented without consuming substantial extra resources through incorporation into an existing patient pathway.
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Genotipo , Diagnóstico Prenatal , Isoinmunización Rh , Sistema del Grupo Sanguíneo Rh-Hr/genética , Adulto , Estudios Transversales , Inglaterra , Femenino , Humanos , Embarazo , Isoinmunización Rh/diagnóstico , Isoinmunización Rh/genéticaRESUMEN
Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.
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Ebolavirus/química , Proteínas del Envoltorio Viral/química , Proteínas Virales de Fusión/química , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/virología , Ebolavirus/fisiología , Dominios Proteicos , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales de Fusión/metabolismo , Internalización del VirusRESUMEN
PURPOSE: To evaluate the safety and efficacy of yttrium-90 transarterial radioembolization (TARE) for the treatment of unresectable, chemotherapy-refractory intrahepatic cholangiocarcinoma (ICC). METHODS: A prospective, observational study was carried out in 10 centers between 2013 and 2017. TARE plus standard care was delivered to patients with unresectable, chemotherapy-refractory or chemotherapy-intolerant ICC. Primary outcome was overall survival. Secondary outcomes included safety, progression-free survival (PFS), and liver-specific progression-free survival (LPFS). RESULTS: Sixty-one patients were treated with TARE. Patients were 53% male; median age was 64 years; 91% had performance status 0/1; 92% had received prior chemotherapy; and 59% had no extrahepatic disease. Median follow-up was 13.9 months (95% confidence interval [CI], 9.6-18.1). Overall survival was 8.7 months (95% CI, 5.3-12.1), and 37% of patients survived to 12 months. PFS was 2.8 months (95% CI, 2.6-3.1), and LPFS was 3.1 months (95% CI, 1.3-4.8). One severe complication (abdominal pain) occurred at the time of the TARE procedure. Thirty patients experienced a total of 49 adverse events, of which 8% were grade ≥3; most common were grade 1-2 fatigue and abdominal pain. A total of 77 abnormal laboratory value events were recorded, of which 4% were grade ≥3. CONCLUSIONS: Patients with advanced ICC have limited therapeutic options and a poor prognosis. This prospective study examined the survival of patients with unresectable, chemotherapy-refractory primary ICC treated with TARE in real-world practice. The results demonstrate that this treatment merits further investigation in this patient cohort in a larger study, including collection of patient-reported outcomes.
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Neoplasias de los Conductos Biliares/radioterapia , Colangiocarcinoma/radioterapia , Resistencia a Antineoplásicos , Embolización Terapéutica/métodos , Radiofármacos/administración & dosificación , Radioisótopos de Itrio/administración & dosificación , Anciano , Neoplasias de los Conductos Biliares/diagnóstico por imagen , Neoplasias de los Conductos Biliares/mortalidad , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/diagnóstico por imagen , Colangiocarcinoma/mortalidad , Colangiocarcinoma/patología , Progresión de la Enfermedad , Embolización Terapéutica/efectos adversos , Embolización Terapéutica/mortalidad , Inglaterra , Femenino , Humanos , Masculino , Persona de Mediana Edad , Supervivencia sin Progresión , Estudios Prospectivos , Radiofármacos/efectos adversos , Factores de Riesgo , Factores de Tiempo , Radioisótopos de Itrio/efectos adversosRESUMEN
Background: A need to develop therapeutics to treat Ebola virus disease patients in remote and resource-challenged settings remains in the wake of the 2013-2016 epidemic in West Africa. Toward this goal, we screened drugs under consideration as treatment options and other drugs of interest, most being small molecules approved by the Food and Drug Administration. Drugs demonstrating in vitro antiviral activity were advanced for evaluation in combinations because of advantages often provided by drug cocktails. Methods: Drugs were screened for blockade of Ebola virus infection in cultured cells. Twelve drugs were tested in all (78 pair-wise) combinations, and 3 were tested in a subset of combinations. Results: Multiple synergistic drug pairs emerged, with the majority comprising 2 entry inhibitors. For the pairs of entry inhibitors studied, synergy was demonstrated at the level of virus entry into host cells. Highly synergistic pairs included aripiprazole/piperacetazine, sertraline/toremifene, sertraline/bepridil, and amodiaquine/clomiphene. Conclusions: Our study shows the feasibility of identifying pairs of approved drugs that synergistically block Ebola virus infection in cell cultures. We discuss our findings in terms of the theoretic ability of these or alternate combinations to reach therapeutic levels. Future research will assess selected combinations in small-animal models of Ebola virus disease.
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Antivirales/administración & dosificación , Ebolavirus/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Chlorocebus aethiops , Aprobación de Drogas , Sinergismo Farmacológico , Quimioterapia Combinada , Células Vero , Virión/efectos de los fármacos , Internalización del Virus/efectos de los fármacosRESUMEN
Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years, a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion-triggering mechanisms. A key take-home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion.
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Endosomas/virología , Internalización del Virus , Virus/metabolismo , Animales , Humanos , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Virus/patogenicidadRESUMEN
UNLABELLED: The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) cause significant morbidity and morality. There is currently no approved therapeutic for highly pathogenic coronaviruses, even as MERS-CoV is spreading throughout the Middle East. We previously screened a library of FDA-approved drugs for inhibitors of coronavirus replication in which we identified Abelson (Abl) kinase inhibitors, including the anticancer drug imatinib, as inhibitors of both SARS-CoV and MERS-CoV in vitro Here we show that the anti-CoV activity of imatinib occurs at the early stages of infection, after internalization and endosomal trafficking, by inhibiting fusion of the virions at the endosomal membrane. We specifically identified the imatinib target, Abelson tyrosine-protein kinase 2 (Abl2), as required for efficient SARS-CoV and MERS-CoV replication in vitro These data demonstrate that specific approved drugs can be characterized in vitro for their anticoronavirus activity and used to identify host proteins required for coronavirus replication. This type of study is an important step in the repurposing of approved drugs for treatment of emerging coronaviruses. IMPORTANCE: Both SARS-CoV and MERS-CoV are zoonotic infections, with bats as the primary source. The 2003 SARS-CoV outbreak began in Guangdong Province in China and spread to humans via civet cats and raccoon dogs in the wet markets before spreading to 37 countries. The virus caused 8,096 confirmed cases of SARS and 774 deaths (a case fatality rate of â¼10%). The MERS-CoV outbreak began in Saudi Arabia and has spread to 27 countries. MERS-CoV is believed to have emerged from bats and passed into humans via camels. The ongoing outbreak of MERS-CoV has resulted in 1,791 cases of MERS and 640 deaths (a case fatality rate of 36%). The emergence of SARS-CoV and MERS-CoV provides evidence that coronaviruses are currently spreading from zoonotic sources and can be highly pathogenic, causing serious morbidity and mortality in humans. Treatment of SARS-CoV and MERS-CoV infection is limited to providing supportive therapy consistent with any serious lung disease, as no specific drugs have been approved as therapeutics. Highly pathogenic coronaviruses are rare and appear to emerge and disappear within just a few years. Currently, MERS-CoV is still spreading, as new infections continue to be reported. The outbreaks of SARS-CoV and MERS-CoV and the continuing diagnosis of new MERS cases highlight the need for finding therapeutics for these diseases and potential future coronavirus outbreaks. Screening FDA-approved drugs streamlines the pipeline for this process, as these drugs have already been tested for safety in humans.
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Antivirales/farmacología , Mesilato de Imatinib/farmacología , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Animales , Línea Celular , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/fisiologíaRESUMEN
Ebolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two-pore Ca(2+) channel 2 (TPC2), a newly identified ebolavirus entry factor. Here, using live cell imaging, we obtained evidence that in contrast to the new model, ebolavirus enters cells through endolysosomes that contain both NPC1 and TPC2.
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Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Ebolavirus/fisiología , Endosomas/virología , Glicoproteínas de Membrana/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Internalización del Virus , Animales , Línea Celular , Chlorocebus aethiops , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microscopía , Modelos Biológicos , Proteína Niemann-Pick C1RESUMEN
UNLABELLED: ZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1(+) endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env. IMPORTANCE: The recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed-embedded in the membrane and present at high density-on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Ebolavirus/inmunología , Ebolavirus/fisiología , Proteínas del Envoltorio Viral/inmunología , Internalización del Virus/efectos de los fármacos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Tomografía con Microscopio Electrónico , Unión Proteica , Proteínas del Envoltorio Viral/metabolismo , Virosomas/inmunología , Virosomas/metabolismoRESUMEN
Lipid rafts in plasma membranes have emerged as possible platforms for the entry of HIV and other viruses into cells. However, little is known about how lipid phase heterogeneity contributes to viral entry because of the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T cell membranes and found that raft-like liquid-ordered (Lo-phase) lipid domains were necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion were low in homogeneous liquid-disordered (Ld-phase) and Lo-phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo-Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld-phase vesicles proceeded only to hemifusion. Thus, we propose that edges but not areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion.
Asunto(s)
Membrana Celular/metabolismo , Colesterol/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Fusión de Membrana , Microdominios de Membrana/metabolismo , Células HEK293 , VIH/fisiología , Humanos , Membrana Dobles de Lípidos/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Linfocitos T/metabolismo , Liposomas Unilamelares , Virión/fisiologíaRESUMEN
UNLABELLED: Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1(+) LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. IMPORTANCE: Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1(+) LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1(+) LE/Lys, as a therapeutic target for SARS and EBOV.