RESUMEN
Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.
Asunto(s)
Encéfalo/citología , Técnicas de Cultivo de Célula , Modelos Biológicos , Organoides , Virus Zika/fisiología , Reactores Biológicos , Técnicas de Cultivo de Célula/economía , Embrión de Mamíferos , Desarrollo Embrionario , Humanos , Células Madre Pluripotentes Inducidas , Neurogénesis , Neuronas/citología , Organoides/virología , Infección por el Virus Zika/fisiopatología , Infección por el Virus Zika/virologíaRESUMEN
Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen identified the approved anthelminthic drug niclosamide as a small molecule inhibitor of Zika virus infection. However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medication. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties. Other modifications in the anilide region with 2'-OMe and 2'-H substitutions were also advantageous. We found that the 4'-NO2 substituent can be replaced with a 4'-CN or 4'-CF3 substituents. Together, these modifications provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic strategies targeting the COVID-19 pandemic.
Asunto(s)
Antivirales/farmacología , Niclosamida/análogos & derivados , Niclosamida/farmacología , SARS-CoV-2/efectos de los fármacos , Virus Zika/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/metabolismo , Sitios de Unión , Chlorocebus aethiops , Estabilidad de Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Niclosamida/metabolismo , Unión Proteica , Ratas , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Relación Estructura-Actividad , Células Vero , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.
Asunto(s)
Daño del ADN , Células-Madre Neurales/metabolismo , Células-Madre Neurales/virología , Replicación Viral , Infección por el Virus Zika/genética , Infección por el Virus Zika/virología , Virus Zika/fisiología , Biomarcadores , Ciclo Celular , Línea Celular , Interacciones Huésped-Patógeno/genética , Humanos , Modelos BiológicosRESUMEN
Zika virus (ZIKV) infection represents a significant threat to the global health system, and the search for efficient antivirals to ZIKV remains necessary and urgent. In this study, we extended the exploration of our previously discovered scaffold of 1H-pyrrolo[1,2-c]imidazol-1-one and revealed that two trans isomers of compounds 2 and 7 and one mixture with major trans isomer of compound 3 as novel tetrahydroquinoline-fused imidazolone derivatives are active against ZIKV infection but they are not virucidal. Western Blot and ELISA analyses of ZIKV NS5 and NS1 further demonstrate that compounds of (±)-2, (±)-3 and (±)-7 act as effective agents against ZIKV infection. We show that the N10's basicity is not the basic requirement for these compounds' antiviral activity in the current work. Importantly, tuning of some pharmacophores including substituents at arene can generate promising candidates for anti-ZIKV agents.
Asunto(s)
Antivirales/farmacología , Infección por el Virus Zika/tratamiento farmacológico , Virus Zika/efectos de los fármacos , Animales , Antivirales/síntesis química , Antivirales/química , Células Cultivadas , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Células Vero , Proteínas no Estructurales Virales/análisis , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Infección por el Virus Zika/metabolismoRESUMEN
Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses that cause widespread, acute febrile illnesses, notably microcephaly for fetuses of infected pregnant women. Detecting the viral cause of these illnesses is paramount to determine risks to patients, counsel pregnant women, and help fight outbreaks. A combined diagnostic algorithm for ZIKV and DENV requires Reverse transcription polymerase chain reaction (RT-PCR) and IgM antibody detection. RT-PCR differentiates between DENV and ZIKV infections during the acute phases of infection, but differentiation based on IgM antibodies is currently nearly impossible in endemic areas. We have developed a ZIKV/DENV protein array and tested it with serum samples collected from ZIKV- and DENV-infected patients and healthy subjects in Puerto Rico. Our analyses reveal a biomarker panel that are capable of discriminating ZIKV and DENV infections with high accuracy, including Capsid protein from African ZIKV strain MR766, and other 5 pair of proteins (NS1, NS2A, NS3, NS4B and NS5) from ZIKV and DENV respectively. Both sensitivity and specificity of the test for ZIKV from DENV are around 90%. We propose that the ZIKV/DENV protein array will be used in future studies to discriminate patients infected with ZIKV from DENV.
Asunto(s)
Dengue/diagnóstico , Proteínas Virales/sangre , Infección por el Virus Zika/diagnóstico , Biomarcadores/sangre , ADN Complementario/genética , ADN Viral/sangre , Dengue/sangre , Virus del Dengue/genética , Humanos , Inmunoglobulina M/sangre , Análisis por Matrices de Proteínas , Virus Zika/genética , Infección por el Virus Zika/sangreRESUMEN
Zika virus (ZIKV) infection causes microcephaly and has been linked to other brain abnormalities. How ZIKV impairs brain development and function is unclear. Here we systematically profiled transcriptomes of human neural progenitor cells exposed to Asian ZIKVC, African ZIKVM, and dengue virus (DENV). In contrast to the robust global transcriptome changes induced by DENV, ZIKV has a more selective and larger impact on expression of genes involved in DNA replication and repair. While overall expression profiles are similar, ZIKVC, but not ZIKVM, induces upregulation of viral response genes and TP53. P53 inhibitors can block the apoptosis induced by both ZIKVC and ZIKVM in hNPCs, with higher potency against ZIKVC-induced apoptosis. Our analyses reveal virus- and strain-specific molecular signatures associated with ZIKV infection. These datasets will help to investigate ZIKV-host interactions and identify neurovirulence determinants of ZIKV.
Asunto(s)
Corteza Cerebral/citología , Perfilación de la Expresión Génica , Células-Madre Neurales/metabolismo , Células-Madre Neurales/virología , Infección por el Virus Zika/genética , Virus Zika/fisiología , Muerte Celular/genética , Línea Celular , Reparación del ADN/genética , Replicación del ADN/genética , Virus del Dengue/fisiología , Humanos , Transducción de Señal/genética , Especificidad de la Especie , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/genética , Infección por el Virus Zika/virologíaRESUMEN
UNLABELLED: Individuals chronically infected with hepatitis C virus (HCV) commonly exhibit hepatic intracellular lipid accumulation, termed steatosis. HCV infection perturbs host lipid metabolism through both cellular and virus-induced mechanisms, with the viral core protein playing an important role in steatosis development. We have recently identified a liver protein, the cell death-inducing DFFA-like effector B (CIDEB), as an HCV entry host dependence factor that is downregulated by HCV infection in a cell culture model. In this study, we investigated the biological significance and molecular mechanism of this downregulation. HCV infection in a mouse model downregulated CIDEB in the liver tissue, and knockout of the CIDEB gene in a hepatoma cell line results in multiple aspects of lipid dysregulation that can contribute to hepatic steatosis, including reduced triglyceride secretion, lower lipidation of very-low-density lipoproteins, and increased lipid droplet (LD) stability. The potential link between CIDEB downregulation and steatosis is further supported by the requirement of the HCV core and its LD localization for CIDEB downregulation, which utilize a proteolytic cleavage event that is independent of the cellular proteasomal degradation of CIDEB. IMPORTANCE: Our data demonstrate that HCV infection of human hepatocytesin vitroandin vivoresults in CIDEB downregulation via a proteolytic cleavage event. Reduction of CIDEB protein levels by HCV or gene editing, in turn, leads to multiple aspects of lipid dysregulation, including LD stabilization. Consequently, CIDEB downregulation may contribute to HCV-induced hepatic steatosis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Hígado Graso/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Animales , Muerte Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo , Hígado Graso/virología , Hepatitis C/virología , Humanos , Lípidos , Ratones , Proteolisis , Ubiquitina/metabolismoRESUMEN
Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types.
Asunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Agregación Celular , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Hepatocitos/fisiología , Células Madre Embrionarias Humanas/fisiología , Humanos , Neuronas Motoras/fisiología , Miocitos Cardíacos/fisiología , Esferoides Celulares/fisiologíaRESUMEN
DHX9 is an ATP-dependent DEXH box helicase with a multitude of cellular functions. Its ability to unwind both DNA and RNA, as well as aberrant, noncanonical polynucleotide structures, has implicated it in transcriptional and translational regulation, DNA replication and repair, and maintenance of genome stability. We report that loss of DHX9 in primary human fibroblasts results in premature senescence, a state of irreversible growth arrest. This is accompanied by morphological defects, elevation of senescence-associated ß-galactosidase levels, and changes in gene expression closely resembling those encountered during replicative (telomere-dependent) senescence. Activation of the p53 signaling pathway was found to be essential to this process. ChIP analysis and investigation of nascent DNA levels revealed that DHX9 is associated with origins of replication and that its suppression leads to a reduction of DNA replication. Our results demonstrate an essential role of DHX9 in DNA replication and normal cell cycle progression.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Senescencia Celular/fisiología , ARN Helicasas DEAD-box/metabolismo , Replicación del ADN/fisiología , Fibroblastos/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/metabolismo , ARN Helicasas DEAD-box/genética , Diploidia , Fibroblastos/citología , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
UNLABELLED: The molecular mechanism of the hepatic tropism of hepatitis C virus (HCV) remains incompletely defined. In vitro hepatic differentiation of pluripotent stem cells produces hepatocyte-like cells (HLCs) permissive for HCV infection, providing an opportunity for studying liver development and host determinants of HCV susceptibility. We previously identified the transition stage of HCV permissiveness and now investigate whether a host protein whose expression is induced during this transition stage is important for HCV infection. We suppressed the expression of a liver-specific protein, cell death-inducing DFFA-like effector b (CIDEB), and performed hepatocyte function and HCV infection assays. We also used a variety of cell-based assays to dissect the specific step of the HCV life cycle that potentially requires CIDEB function. We found CIDEB to be an essential cofactor for HCV entry into hepatocytes. Genetic interference with CIDEB in stem cells followed by hepatic differentiation leads to HLCs that are refractory to HCV infection, and infection time course experiments revealed that CIDEB functions in a late step of HCV entry, possibly to facilitate membrane fusion. The role of CIDEB in mediating HCV entry is distinct from those of the well-established receptors, as it is not required for HCV pseudoparticle entry. Finally, HCV infection effectively downregulates CIDEB protein through a posttranscriptional mechanism. IMPORTANCE: This study identifies a hepatitis C virus (HCV) entry cofactor that is required for HCV infection of hepatocytes and potentially facilitates membrane fusion between viral and host membranes. CIDEB and its interaction with HCV may open up new avenues of investigation of lipid droplets and viral entry.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Hepacivirus/fisiología , Hepatocitos/virología , Interacciones Huésped-Patógeno , Internalización del Virus , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Técnicas de Silenciamiento del Gen , HumanosRESUMEN
ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3 mimetic to neutralize Bcl-2-like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and thus is a major cause of resistance. We performed a short hairpin RNA (shRNA)-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eµ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival. Several drug-sensitive shRNAs were identified that were selectively depleted in the presence of ABT-737. Of these, 2 independent shRNAs targeting the RNA/DNA helicase Dhx9 were found to sensitize lymphomas to ABT-737 to an extent comparable to control Mcl-1 shRNAs. Although Dhx9 suppression sensitized both mouse and human cells to ABT-737 treatment, it did so without altering MCL-1 levels. Rather, loss of Dhx9 appeared to activate a p53-dependent apoptotic program, through aggravation of replicative stress, which was found to be both necessary and sufficient for the ABT-737-shDhx9 synthetic lethal relationship.
Asunto(s)
Compuestos de Bifenilo/farmacología , ARN Helicasas DEAD-box/genética , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Linfoma/genética , Nitrofenoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , Sulfonamidas/farmacología , Animales , Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/metabolismo , Genes Modificadores , Humanos , Linfoma/patología , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Primary human hepatocytes isolated from patient biopsies represent the most physiologically relevant cell culture model for hepatitis C virus (HCV) infection, but these primary cells are not readily accessible, display individual variability, and are largely refractory to genetic manipulation. Hepatocyte-like cells differentiated from pluripotent stem cells provide an attractive alternative as they not only overcome these shortcomings but can also provide an unlimited source of noncancer cells for both research and cell therapy. Despite its promise, the permissiveness to HCV infection of differentiated human hepatocyte-like cells (DHHs) has not been explored. Here we report a novel infection model based on DHHs derived from human embryonic (hESCs) and induced pluripotent stem cells (iPSCs). DHHs generated in chemically defined media under feeder-free conditions were subjected to infection by both HCV derived in cell culture (HCVcc) and patient-derived virus (HCVser). Pluripotent stem cells and definitive endoderm were not permissive for HCV infection whereas hepatic progenitor cells were persistently infected and secreted infectious particles into culture medium. Permissiveness to infection was correlated with induction of the liver-specific microRNA-122 and modulation of cellular factors that affect HCV replication. RNA interference directed toward essential cellular cofactors in stem cells resulted in HCV-resistant hepatocyte-like cells after differentiation. The ability to infect cultured cells directly with HCV patient serum, to study defined stages of viral permissiveness, and to produce genetically modified cells with desired phenotypes all have broad significance for host-pathogen interactions and cell therapy.
Asunto(s)
Diferenciación Celular , Hepacivirus/fisiología , Hepatitis C/virología , Hepatocitos/virología , Modelos Biológicos , Células Madre Pluripotentes/virología , Línea Celular , Hepatitis C/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Interacciones Huésped-Patógeno/fisiología , Humanos , MicroARNs/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patologíaRESUMEN
Zika virus (ZIKV), a mosquito-borne flavivirus, is prominently associated with microcephaly in babies born to infected mothers as well as Guillain-Barré Syndrome in adults. Each cell type infected by ZIKV-neuronal cells (radial glial cells, neuronal progenitor cells, astrocytes, microglia cells, and glioblastoma stem cells) and non-neuronal cells (primary fibroblasts, epidermal keratinocytes, dendritic cells, monocytes, macrophages, and Sertoli cells)-displays its own characteristic changes to their cell physiology and has various impacts on disease. Here, we provide an in-depth review of the ZIKV life cycle and its cellular targets, and discuss the current knowledge of how infections cause neuropathologies, as well as what approaches researchers are currently taking to further advance such knowledge. A key aspect of ZIKV neuropathogenesis is virus-induced neuronal apoptosis via numerous mechanisms including cell cycle dysregulation, mitochondrial fragmentation, ER stress, and the unfolded protein response. These, in turn, result in the activation of p53-mediated intrinsic cell death pathways. A full spectrum of infection models including stem cells and co-cultures, transwells to simulate blood-tissue barriers, brain-region-specific organoids, and animal models have been developed for ZIKV research.
RESUMEN
Cyclophilin A (CyPA) and its peptidyl-prolyl isomerase (PPIase) activity play an essential role in hepatitis C virus (HCV) replication, and mounting evidence indicates that nonstructural protein 5A (NS5A) is the major target of CyPA. However, neither a consensus CyPA-binding motif nor specific proline substrates that regulate CyPA dependence and sensitivity to cyclophilin inhibitors (CPIs) have been defined to date. We systematically characterized all proline residues in NS5A domain II, low-complexity sequence II (LCS-II), and domain III with both biochemical binding and functional replication assays. A tandem cyclophilin-binding site spanning domain II and LCS-II was identified. The first site contains a consensus sequence motif of AØPXW (where Ø is a hydrophobic residue) that is highly conserved in the majority of the genotypes of HCV (six of seven; the remaining genotype has VØPXW). The second tandem site contains a similar motif, and the ØP sequence is again conserved in six of the seven genotypes. Consistent with the similarity of their sequences, peptides representing the two binding motifs competed for CyPA binding in a spot-binding assay and induced similar chemical shifts when bound to the active site of CyPA. The two prolines (P310 and P341 of Japanese fulminant hepatitis 1 [JFH-1]) contained in these motifs, as well as a conserved tryptophan in the spacer region, were required for CyPA binding, HCV replication, and CPI resistance. Together, these data provide a high-resolution mapping of proline residues important for CyPA binding and identify critical amino acids modulating HCV susceptibility to the clinical CPI Alisporivir.
Asunto(s)
Secuencia Conservada , Ciclofilina A/metabolismo , Ciclosporina/farmacología , Hepacivirus/efectos de los fármacos , Secuencias Repetidas en Tándem , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Línea Celular , Farmacorresistencia Viral/genética , Genotipo , Humanos , Datos de Secuencia Molecular , Dominios Proteicos Ricos en Prolina , Unión Proteica , Conformación Proteica , Proteínas no Estructurales Virales/genéticaRESUMEN
Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.
Asunto(s)
Ciclofilina A/antagonistas & inhibidores , Ciclosporina/farmacología , Hepacivirus/efectos de los fármacos , Modelos Biológicos , ARN Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus/efectos de los fármacos , Western Blotting , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Hepacivirus/fisiología , Humanos , Inmunoprecipitación , Técnicas In Vitro , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensamble de Virus/fisiologíaRESUMEN
Viruses are known to use virally encoded envelope proteins for cell attachment, which is the very first step of virus infection. In the present study, we have obtained substantial evidence demonstrating that hepatitis C virus (HCV) uses the cellular protein apolipoprotein E (apoE) for its attachment to cells. An apoE-specific monoclonal antibody was able to efficiently block HCV attachment to the hepatoma cell line Huh-7.5 as well as primary human hepatocytes. After HCV bound to cells, however, anti-apoE antibody was unable to inhibit virus infection. Conversely, the HCV E2-specific monoclonal antibody CBH5 did not affect HCV attachment but potently inhibited HCV entry. Similarly, small interfering RNA-mediated knockdown of the key HCV receptor/coreceptor molecules CD81, claudin-1, low-density lipoprotein receptor (LDLr), occludin, and SR-BI did not affect HCV attachment but efficiently suppressed HCV infection, suggesting their important roles in HCV infection at postattachment steps. Strikingly, removal of heparan sulfate from the cell surface by treatment with heparinase blocked HCV attachment. Likewise, substitutions of the positively charged amino acids with neutral or negatively charged residues in the receptor-binding region of apoE resulted in a reduction of apoE-mediating HCV infection. More importantly, mutations of the arginine and lysine to alanine or glutamic acid in the receptor-binding region ablated the heparin-binding activity of apoE, as determined by an in vitro heparin pulldown assay. HCV attachment could also be inhibited by a synthetic peptide derived from the apoE receptor-binding region. Collectively, these findings demonstrate that apoE mediates HCV attachment through specific interactions with cell surface heparan sulfate.
Asunto(s)
Apolipoproteínas E/metabolismo , Hepacivirus/fisiología , Heparitina Sulfato/metabolismo , Hepatocitos/virología , Acoplamiento Viral , Apolipoproteínas E/genética , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Puntual , Unión Proteica , Receptores Virales/antagonistas & inhibidores , Receptores Virales/metabolismoRESUMEN
Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.
Asunto(s)
Ciclofilina A/metabolismo , Ciclosporina/farmacología , Farmacorresistencia Viral , Regulación Viral de la Expresión Génica , Hepacivirus/efectos de los fármacos , Hepacivirus/metabolismo , Hepatitis C/tratamiento farmacológico , Secuencia de Aminoácidos , Western Blotting , Línea Celular , Dicroismo Circular , Ciclofilina A/genética , Ensayo de Inmunoadsorción Enzimática , Hepacivirus/genética , Hepatitis C/genética , Hepatitis C/virología , Humanos , Técnicas para Inmunoenzimas , Inmunosupresores/farmacología , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Conformación Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , ARN Viral/genética , Replicón/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The blood-brain barrier is a tissue structure that modulates the selective entry of molecules into the brain compartment. This barrier offers protection to the brain microenvironment from toxins or any fluctuations in the composition of the blood plasma via a layer of endothelial cells connected by tight junctions and supported by pericytes and astrocytes. Disruption of the barrier can be either a cause or a consequence of central nervous system pathogenesis. Therefore, research based on understanding the structure, function, and the mechanisms of breaching the blood-brain barrier is of primary interest for diverse disciplines including drug discovery, brain pathology, and infectious disease. The following protocol describes a detailed differentiation method that uses defined serum components during stem cell culture to deliver cellular cues in order to drive the cells towards brain endothelial cell lineage. This method can be used to obtain reproducible and scalable cultures of brain microvascular endothelial cells with barrier characteristics and functionality. These endothelial cells can also be stored long term or shipped frozen.
Asunto(s)
Barrera Hematoencefálica , Células Endoteliales , Astrocitos , Encéfalo , Diferenciación Celular/fisiología , Pericitos , Uniones EstrechasRESUMEN
Background: Recently, the in vitro blood brain barrier (BBB) models derived from human pluripotent stem cells have been given extensive attention in therapeutics due to the implications it has with the health of the central nervous system. It is essential to create an accurate BBB model in vitro in order to better understand the properties of the BBB and how it can respond to inflammatory stimulation and be passed by targeted or non-targeted cell therapeutics, more specifically extracellular vesicles. Methods: Brain-specific pericytes (iPCs) were differentiated from iPSK3 cells using dual SMAD signaling inhibitors and Wnt activation plus fibroblast growth factor 2 (FGF-2). The derived cells were characterized by immunostaining, flow cytometry and RT-PCR. In parallel, blood vessels organoids were derived using Wnt activation, BMP4, FGF2, VEGF and SB431542. The organoids were replated and treated with retinoic acid to enhance the blood brain barrier (BBB) features in the differentiated brain endothelial cells (iECs). Co-culture was performed for the iPCs and iECs in transwell system and 3-D microfluidics channels. Results: The derived iPCs expressed common markers PDGFRb and NG2, as well as brain-specific genes FOXF2, ABCC9, KCNJ8, and ZIC1. The derived iECs expressed common endothelial cell markers CD31, VE-cadherin, as well as BBB-associated genes BRCP, GLUT-1, PGP, ABCC1, OCLN, SLC2A1. The co-culture of the two cell types responded to the stimulation of amyloid ß42 oligomers by the upregulation of expression of TNFa, IL6, NFKB, Casp3, SOD2 and TP53. The co-culture also showed the property of trans-endothelial electrical resistance. The proof-of-concept vascularization strategy was demonstrated in a 3-D microfluidics-based device. Conclusion: The derived iPCs and iECs have brain-specific properties and the co-culture of iPCs and iECs provides an in vitro BBB model that show inflammatory response. This study has significance in establishing micro-physiological systems for neurological disease modeling and drug screening.
RESUMEN
Physiological blood-tissue barriers play a critical role in separating the circulation from immune-privileged sites and denying access to blood-borne viruses. The mechanism of virus restriction by these barriers is poorly understood. We utilize induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (iBMECs) to study virus-blood-brain barrier (BBB) interactions. These iPSC-derived cells faithfully recapitulate a striking difference in in vivo neuroinvasion by two alphavirus isolates and are selectively permissive to neurotropic flaviviruses. A model of cocultured iBMECs and astrocytes exhibits high transendothelial electrical resistance and blocks non-neurotropic flaviviruses from getting across the barrier. We find that iBMECs constitutively express an interferon-induced gene, IFITM1, which preferentially restricts the replication of non-neurotropic flaviviruses. Barrier cells from blood-testis and blood-retinal barriers also constitutively express IFITMs that contribute to the viral resistance. Our application of a renewable human iPSC-based model for studying virus-BBB interactions reveals that intrinsic immunity at the barriers contributes to virus exclusion.