RESUMEN

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions.

RESUMEN

The Type-I interferon (IFN-I) response is the major outcome of stimulator of interferon genes (STING) activation in innate cells. STING is more abundantly expressed in adaptive T cells; nevertheless, its intrinsic function in T cells remains unclear. Intriguingly, we previously demonstrated that STING activation in T cells activates widespread IFN-independent activities, which stands in contrast to the well-known STING-mediated IFN response. Here, we have identified that STING activation induces regulatory T cells (Tregs) differentiation independently of IRF3 and IFN. Specifically, the translocation of STING from the endoplasmic reticulum to the Golgi activates mitogen-activated protein kinase (MAPK) activity, which subsequently triggers transcription factor cAMP response element-binding protein (CREB) activation. The activation of the STING-MAPK-CREB signaling pathway induces the expression of many cytokine genes, including interleukin-2 (IL-2) and transforming growth factor-beta 2 (TGF-ß2), to promote the Treg differentiation. Genetic knockdown of MAPK p38 or pharmacological inhibition of MAPK p38 or CREB markedly inhibits STING-mediated Treg differentiation. Administration of the STING agonist also promotes Treg differentiation in mice. In the Trex1-/- autoimmune disease mouse model, we demonstrate that intrinsic STING activation in CD4+ T cells can drive Treg differentiation, potentially counterbalancing the autoimmunity associated with Trex1 deficiency. Thus, STING-MAPK-CREB represents an IFN-independent signaling axis of STING that may have profound effects on T cell effector function and adaptive immunity.

Asunto(s)

Diferenciación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas de la Membrana , Linfocitos T Reguladores , Animales , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ratones , Transducción de Señal , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Transporte de Proteínas , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/genética , Ratones Noqueados , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.

Asunto(s)

Hepatitis B Crónica , Neoplasias Hepáticas , Humanos , Virus de la Hepatitis B/genética , Antivirales/farmacología , Antivirales/uso terapéutico , ARN , Hepatitis B Crónica/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Replicación Viral

RESUMEN

Inherited genetic variations in the melanocortin-1 receptor (MC1R) responsible for human red hair color (RHC) variants are associated with impaired DNA damage repair and increased melanoma risk. MC1R signaling is critically dependent on palmitoylation, primarily mediated by the protein acyltransferase zinc finger DHHC-type palmitoyltransferase 13 (ZDHHC13). A better understanding of how ZDHHC13 is physiologically activated could help identify approaches to prevent melanomagenesis in redheads. Here, we report that AMP-activated protein kinase (AMPK) phosphorylates ZDHHC13 at S208 to strengthen the interaction between ZDHHC13 and MC1R-RHC, leading to enhanced MC1R palmitoylation in redheads. Consequently, phosphorylation of ZDHHC13 by AMPK increased MC1R-RHC downstream signaling. AMPK activation and MC1R palmitoylation repressed UVB-induced transformation of human melanocytes in vitro and delayed melanomagenesis in vivo in C57BL/6J-MC1R-RHC mice. The importance of AMPK to MC1R signaling was validated in human melanomas where AMPK upregulation correlated with expression of factors downstream from MC1R signaling and with prolonged patient survival. These findings suggest AMPK activation as a promising strategy to reduce melanoma risk, especially for individuals with red hair. SIGNIFICANCE: Phosphorylation of ZDHHC13 by AMPK at S208 promotes MC1R activation and suppresses melanocyte transformation, indicating activation of AMPK as a potential approach to prevent melanoma in people with red hair.

Asunto(s)

Proteínas Quinasas Activadas por AMP , Transformación Celular Neoplásica , Melanoma , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Activación Enzimática , Fosforilación , Lipoilación , Melanocitos/enzimología , Melanocitos/efectos de la radiación , Humanos , Animales , Ratones , Melanoma/genética , Rayos Ultravioleta , Regulación de la Expresión Génica/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/efectos de la radiación

RESUMEN

BACKGROUND & AIMS: A number of genetic polymorphisms have been associated with susceptibility to or protection against non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain unknown. Here, we focused on the rs738409 C>G single nucleotide polymorphism (SNP), which produces the I148M variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3) and is strongly associated with NAFLD. METHODS: To enable mechanistic dissection, we developed a human pluripotent stem cell (hPSC)-derived multicellular liver culture by incorporating hPSC-derived hepatocytes, hepatic stellate cells, and macrophages. We first applied this liver culture to model NAFLD by utilising a lipotoxic milieu reflecting the circulating levels of disease risk factors in affected individuals. We then created an isogenic pair of liver cultures differing only at rs738049 and compared NAFLD phenotype development. RESULTS: Our hPSC-derived liver culture recapitulated many key characteristics of NAFLD development and progression including lipid accumulation and oxidative stress, inflammatory response, and stellate cell activation. Under the lipotoxic conditions, the I148M variant caused the enhanced development of NAFLD phenotypes. These differences were associated with elevated IL-6/signal transducer and activator of transcription 3 (STAT3) activity in liver cultures, consistent with transcriptomic data of liver biopsies from individuals carrying the rs738409 SNP. Dampening IL-6/STAT3 activity alleviated the I148M-mediated susceptibility to NAFLD, whereas boosting it in wild-type liver cultures enhanced NAFLD development. Finally, we attributed this elevated IL-6/STAT3 activity in liver cultures carrying the rs738409 SNP to increased NF-κB activity. CONCLUSIONS: Our study thus reveals a potential causal link between elevated IL-6/STAT3 activity and 148M-mediated susceptibility to NAFLD. IMPACT AND IMPLICATIONS: An increasing number of genetic variants manifest in non-alcoholic fatty liver disease (NAFLD) development and progression; however, the underlying mechanisms remain elusive. To study these variants in human-relevant systems, we developed an induced pluripotent stem cell-derived multicellular liver culture and focused on a common genetic variant (i.e. rs738409 in PNPLA3). Our findings not only provide mechanistic insight, but also a potential therapeutic strategy for NAFLD driven by this genetic variant in PNPLA3. Our liver culture is therefore a useful platform for exploring genetic variants in NAFLD development.

Asunto(s)

Enfermedad del Hígado Graso no Alcohólico , Fosfolipasas A2 Calcio-Independiente , Humanos , Predisposición Genética a la Enfermedad , Interleucina-6/genética , Interleucina-6/metabolismo , Hígado/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Fosfolipasas A2 Calcio-Independiente/genética , Polimorfismo de Nucleótido Simple , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo

RESUMEN

Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are valuable tools to study liver biology. HLCs, however, lack certain key in vivo characteristics relevant to their physiological function. One such characteristic is cellular polarity, which is critical to hepatocyte counter-current flow systems involving canalicular bile secretion and sinusoidal secretion of large quantities of serum proteins into blood. Model systems using non-polarized hepatocytes, therefore, cannot recapitulate this physiological function of hepatocytes. Here, we describe a stepwise protocol to generate hPSC-derived polarized HLCs (pol-HLCs), which feature clearly defined basolateral and apical membranes separated by tight junctions. Pol-HLCs not only display many hepatic functions but are also capable of directional cargo secretion, mimicking the counter-current flow systems. We describe protocols for stem cell culture maintenance and for differentiating hPSCs into pol-HLCs. In addition, we describe protocols to assay the pol-HLCs for basic hepatic functions and polarized hepatic characteristics. Once successfully differentiated, these pol-HLCs can be used as an in vitro model system to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism. The establishment of hepatic polarity from non-polarized hPSCs also provides a useful tool to study the development and maintenance of hepatic polarity. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Maintenance of hPSCs Basic Protocol 2: Differentiation of hPSCs to pol-HLCs Basic Protocol 3: Assaying pol-HLCs for basic hepatic functions Support Protocol 1: Assessment of pol-HLC monolayer tightness Support Protocol 2: Assessment of pol-HLC polarity.

Asunto(s)

Hepatocitos , Células Madre Pluripotentes , Técnicas de Cultivo de Célula , Diferenciación Celular , Humanos , Hígado

RESUMEN

Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.

Asunto(s)

Adenosina Desaminasa/genética , Regulación de la Expresión Génica , Isoformas de Proteínas/genética , Edición de ARN , Proteínas de Unión al ARN/genética , Enfermedades Autoinmunes del Sistema Nervioso/genética , Susceptibilidad a Enfermedades , Técnicas de Inactivación de Genes , Predisposición Genética a la Enfermedad , Humanos , Malformaciones del Sistema Nervioso/genética , Trastornos de la Pigmentación/congénito , Trastornos de la Pigmentación/genética

RESUMEN

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.

Asunto(s)

Infecciones por Flavivirus/genética , Flavivirus/fisiología , Proteínas de la Membrana/metabolismo , Animales , Pueblo Asiatico/genética , Autofagia , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Sistemas CRISPR-Cas , Línea Celular , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/metabolismo , Infecciones por Flavivirus/virología , Técnicas de Inactivación de Genes , Estudio de Asociación del Genoma Completo , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , SARS-CoV-2/fisiología , Replicación Viral , Virus de la Fiebre Amarilla/fisiología , Virus Zika/fisiología

RESUMEN

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication. HIGHLIGHTS: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.

RESUMEN

Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease.

Asunto(s)

Cisteína Endopeptidasas/metabolismo , Infecciones por Enterovirus/virología , Enterovirus/patogenicidad , Replicación Viral/fisiología , Antivirales/metabolismo , Enterovirus/metabolismo , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteolisis , Proteínas Virales/metabolismo

RESUMEN

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Polaridad Celular , Hepatocitos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Antivirales/farmacología , Diferenciación Celular , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Interacciones Farmacológicas , Virus de la Hepatitis A Humana/fisiología , Virus de la Hepatitis E/fisiología , Hepatocitos/ultraestructura , Hepatocitos/virología , Humanos , Hígado/citología , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Microscopía Electrónica de Transmisión , Prueba de Estudio Conceptual , Virión/metabolismo , Liberación del Virus , Replicación Viral

RESUMEN

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and ß-thalassemia1. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development2-7. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.

Asunto(s)

Hemoglobinas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Adulto , Animales , Sitios de Unión , Células Cultivadas , Células Eritroides/metabolismo , Eritropoyesis/genética , Regulación de la Expresión Génica , Hemoglobinas/genética , Humanos , Recién Nacido , MicroARNs/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Ribosómico 18S/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Represoras/metabolismo , Ribosomas/genética , Ribosomas/metabolismo

RESUMEN

Given the unprecedented scale of the recent Ebola and Zika viral epidemics, it is crucial to understand the biology of host factors with broad antiviral action in order to develop novel therapeutic approaches. Here, we look into one such factor: zinc finger antiviral protein (ZAP) inhibits a variety of RNA and DNA viruses. Alternative splicing results in two isoforms that differ at their C termini: ZAPL (long) encodes a poly(ADP-ribose) polymerase (PARP)-like domain that is missing in ZAPS (short). Previously, it has been shown that ZAPL is more antiviral than ZAPS, while the latter is more induced by interferon (IFN). In this study, we discovered and confirmed the expression of two additional splice variants of human ZAP: ZAPXL (extralong) and ZAPM (medium). We also found two haplotypes of human ZAP. Since ZAPL and ZAPS have differential activities, we hypothesize that all four ZAP isoforms have evolved to mediate distinct antiviral and/or cellular functions. By taking a gene-knockout-and-reconstitution approach, we have characterized the antiviral, translational inhibition, and IFN activation activities of individual ZAP isoforms. Our work demonstrates that ZAPL and ZAPXL are more active against alphaviruses and hepatitis B virus (HBV) than ZAPS and ZAPM and elucidates the effects of splice variants on the action of a broad-spectrum antiviral factor.IMPORTANCE ZAP is an IFN-induced host factor that can inhibit a wide range of viruses, and there is great interest in fully characterizing its antiviral mechanism. This is the first study that defines the antiviral capacities of individual ZAP isoforms in the absence of endogenous ZAP expression and, hence, cross talk with other isoforms. Our data demonstrate that ZAP is expressed as four different forms: ZAPS, ZAPM, ZAPL, and ZAPXL. The longer ZAP isoforms better inhibit alphaviruses and HBV, while all isoforms equally inhibit Ebola virus transcription and replication. In addition, there is no difference in the abilities of ZAP isoforms to enhance the induction of type I IFN expression. Our results show that the full spectrum of ZAP activities can change depending on the virus target and the relative levels of basal expression and induction by IFN or infection.

Asunto(s)

Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Células A549 , Alphavirus/genética , Empalme Alternativo , Línea Celular , Células HEK293 , Haplotipos , Células HeLa , Virus de la Hepatitis B/genética , Humanos , Isoformas de Proteínas , Empalme del ARN/genética , ARN Viral/genética , Replicación Viral/efectos de los fármacos , Dedos de Zinc

RESUMEN

The erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, was the first hematopoietic niche discovered. The identity of EBI macrophages has thus far remained elusive. Given that Epo is essential for erythropoiesis and that Epor is expressed in numerous nonerythroid cells, we hypothesized that EBI macrophages express Epor so that Epo can act on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis. To test this notion, we used Epor-eGFPcre knockin mouse model. We show that in bone marrow (BM) and fetal liver, a subset of macrophages express Epor-eGFP. Imaging flow cytometry analyses revealed that >90% of native EBIs comprised F4/80+Epor-eGFP+ macrophages. Human fetal liver EBIs also comprised EPOR+ macrophages. Gene expression profiles of BM F4/80+Epor-eGFP+ macrophages suggest a specialized function in supporting erythropoiesis. Molecules known to be important for EBI macrophage function such as Vcam1, CD169, Mertk, and Dnase2α were highly expressed in F4/80+Epor-eGFP+ macrophages compared with F4/80+Epor-eGFP- macrophages. Key molecules involved in iron recycling were also highly expressed in BM F4/80+Epor-eGFP+ macrophages, suggesting that EBI macrophages may provide an iron source for erythropoiesis within this niche. Thus, we have characterized EBI macrophages in mouse and man. Our findings provide important resources for future studies of EBI macrophage function during normal as well as disordered erythropoiesis in hematologic diseases such as thalassemia, polycythemia vera, and myelodysplastic syndromes.

Asunto(s)

Eritroblastos/metabolismo , Perfilación de la Expresión Génica , Macrófagos/metabolismo , Transcriptoma , Animales , Biomarcadores , Biología Computacional/métodos , Eritropoyesis/genética , Expresión Génica , Humanos , Inmunofenotipificación , Ratones , Monocitos/metabolismo , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Nicho de Células Madre/genética , Estrés Fisiológico

RESUMEN

Human-induced pluripotent stem cell-derived hepatocyte-like cells (iHeps) constitute a powerful tool for modeling hepatotropic pathogen infections in cell culture. Meanwhile, CRISPR-Cas9 technology enables precise editing of stem cell genomes to generate patient-specific disease models and thus development of personalized experimental systems. Here we present a detailed stepwise protocol for the differentiation of stem cells to hepatocyte-like cells for HCV studies in cell culture. We also outline the use of an inducible iCRISPR platform for the rapid and efficient modification of host factors of interest to better understand their function during HCV infection.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/citología , Sistemas CRISPR-Cas , Línea Celular , Edición Génica/métodos , Hepacivirus/fisiología , Hepatitis C/genética , Hepatocitos/metabolismo , Interacciones Huésped-Patógeno , Humanos , Células Madre Pluripotentes Inducidas/metabolismo

RESUMEN

Similar to other hepatotropic viruses, hepatitis E virus (HEV) has been notoriously difficult to propagate in cell culture, limiting studies to unravel its biology. Recently, major advances have been made by passaging primary HEV isolates and selecting variants that replicate efficiently in carcinoma cells. These adaptations, however, can alter HEV biology. We have explored human embryonic or induced pluripotent stem cell (hESC/iPSC)-derived hepatocyte-like cells (HLCs) as an alternative to conventional hepatoma and hepatocyte cell culture systems for HEV studies. HLCs are permissive for nonadapted HEV isolate genotypes (gt)1-4 replication and can be readily genetically manipulated. HLCs, therefore, enable studies of pan-genotype HEV biology and will serve as a platform for testing anti-HEV treatments. Finally, we discuss how hepatocyte polarity is likely an important factor in the maturation and spread of infectious HEV particles.

Asunto(s)

Técnicas de Cultivo de Célula , Virus de la Hepatitis E/fisiología , Hepatitis E/virología , Células Madre/fisiología , Adaptación Fisiológica , Carcinoma Hepatocelular/virología , Humanos , Neoplasias Hepáticas/virología , Células Tumorales Cultivadas , Replicación Viral/fisiología

RESUMEN

Stem cells are important for growth and regeneration given their ability to self-renew and differentiate into mature cells. Resistance to certain viral infections has been established as a phenotype of stem cells, a protection in line with their important physiological function. Antiviral resistance is critical to all cells, but it is differentially regulated between stem cells and differentiated cells. Stem cells utilize antiviral RNA interference, interferon-independent repression of endogenous retroviruses and intrinsic expression of antiviral interferon-stimulated genes. Differentiated cells often rely on the interferon-associated protein-based response to induce a local antiviral state. This review outlines the antiviral resistance mechanisms of stem cells and discusses some ideas as to why stem cells and differentiated cells may have evolved to utilize distinct mechanisms.

Asunto(s)

Retrovirus Endógenos/inmunología , ARN Viral/genética , Células Madre/fisiología , Virosis/inmunología , Animales , Antivirales/metabolismo , Diferenciación Celular , Resistencia a la Enfermedad , Represión Epigenética , Humanos , Inmunidad Innata , Interferones/metabolismo , Interferencia de ARN , Transducción de Señal

RESUMEN

Type I interferon (IFN) is produced when host sensors detect foreign nucleic acids, but how sensors differentiate self from nonself nucleic acids, such as double-stranded RNA (dsRNA), is incompletely understood. Mutations in ADAR1, an adenosine-to-inosine editing enzyme of dsRNA, cause Aicardi-Goutières syndrome, an autoinflammatory disorder associated with spontaneous interferon production and neurologic sequelae. We generated ADAR1 knockout human cells to explore ADAR1 substrates and function. ADAR1 primarily edited Alu elements in RNA polymerase II (pol II)-transcribed mRNAs, but not putative pol III-transcribed Alus. During the IFN response, ADAR1 blocked translational shutdown by inhibiting hyperactivation of PKR, a dsRNA sensor. ADAR1 dsRNA binding and catalytic activities were required to fully prevent endogenous RNA from activating PKR. Remarkably, ADAR1 knockout neuronal progenitor cells exhibited MDA5 (dsRNA sensor)-dependent spontaneous interferon production, PKR activation, and cell death. Thus, human ADAR1 regulates sensing of self versus nonself RNA, allowing pathogen detection while avoiding autoinflammation.

Asunto(s)

Adenosina Desaminasa/metabolismo , Elementos Alu , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Biosíntesis de Proteínas , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Muerte Celular/genética , Muerte Celular/inmunología , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/inmunología , Helicasa Inducida por Interferón IFIH1/metabolismo , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Células-Madre Neurales/citología , Células-Madre Neurales/inmunología , Células-Madre Neurales/patología , ARN Polimerasa II/genética , ARN Polimerasa II/inmunología , ARN Polimerasa II/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/inmunología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , eIF-2 Quinasa/genética , eIF-2 Quinasa/inmunología , eIF-2 Quinasa/metabolismo

RESUMEN

BACKGROUND & AIMS: The 4 genotypes of hepatitis E virus (HEV) that infect humans (genotypes 1-4) vary in geographical distribution, transmission, and pathogenesis. Little is known about the properties of HEV or its hosts that contribute to these variations. Primary isolates grow poorly in cell culture; most studies have relied on variants adapted to cancer cell lines, which likely alter virus biology. We investigated the infection and replication of primary isolates of HEV in hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells. METHODS: Using a cell culture-adapted genotype 3 strain and primary isolates of genotypes 1 to 4, we compared viral replication kinetics, sensitivity to drugs, and ability of HEV to activate the innate immune response. We studied HLCs using quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence assay and enzyme-linked immunosorbent assays. We used an embryonic stem cell line that can be induced to express the CRISPR-Cas9 machinery to disrupt the peptidylprolyl isomerase A gene, encoding cyclophilin A (CYPA), a protein reported to inhibit replication of cell culture-adapted HEV. We further modified this line to rescue expression of CYPA before terminal differentiation to HLCs and performed HEV infection studies. RESULTS: HLCs were permissive for infection by nonadapted, primary isolates of HEV genotypes 1 to 4. HEV infection of HLCs induced a replication-dependent type III interferon response. Replication of primary HEV isolates, unlike the cell culture-adapted strain, was not affected by disruption of the peptidylprolyl isomerase A gene or exposure to the CYPA inhibitor cyclosporine A. CONCLUSIONS: Cell culture adaptations alter the replicative capacities of HEV. HLCs offer an improved, physiologically relevant, and genetically tractable system for studying the replication of primary HEV isolates. HLCs could provide a model to aid development of HEV drugs and a system to guide personalized regimens, especially for patients with chronic hepatitis E who have developed resistance to ribavirin.

Asunto(s)

Virus de la Hepatitis E/crecimiento & desarrollo , Hepatocitos/virología , Células Madre Embrionarias Humanas/virología , Células Madre Pluripotentes Inducidas/virología , Replicación Viral , Antivirales/farmacología , Diferenciación Celular , Ciclofilina A/genética , Ciclofilina A/metabolismo , Farmacorresistencia Viral , Genotipo , Células Hep G2 , Virus de la Hepatitis E/efectos de los fármacos , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/inmunología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Interacciones Huésped-Patógeno , Células Madre Embrionarias Humanas/inmunología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunidad Innata , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Fenotipo , ARN Viral/genética , Sofosbuvir/farmacología , Factores de Tiempo , Transfección , Replicación Viral/efectos de los fármacos

RESUMEN

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.

Asunto(s)

Inmunidad Innata , Células Madre Pluripotentes/inmunología , Virosis/inmunología , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Interferones/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Células Madre Pluripotentes/virología , Especificidad de la Especie