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.
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•Pluri-/multipotent stem cells exhibit intrinsic expression of ISGs•Different stem cells express cell-type-specific groups of ISGs•Intrinsically expressed ISGs mediate antiviral resistance ex vivo and in vivo•Dynamic expression of ISGs is conserved across species
Intrinsic expression of interferon-stimulated genes makes stem cells resistant to infections, preserving their pool throughout the organism’s lifespan.
Infection with hepatitis E virus genotype 3 may result in chronic hepatitis in immunocompromised patients. Reduction of immunosuppression or treatment with ribavirin or pegylated interferon-α can ...result in viral clearance. However, safer and more effective treatment options are needed. Here, we show that sofosbuvir inhibits the replication of hepatitis E virus genotype 3 both in subgenomic replicon systems as well as a full-length infectious clone. Moreover, the combination of sofosbuvir and ribavirin results in an additive antiviral effect. Sofosbuvir may be considered as an add-on therapy to ribavirin for the treatment of chronic hepatitis E in immunocompromised patients.
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.
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•ADAR1 edits are found in putative RNA polymerase II (but not III)-derived Alu repeats•ADAR1 prevents translational shutdown during the type I IFN response•ADAR1 dsRNA binding and catalytic activities are required to block PKR activation•ADAR1 KO NPCs exhibit MDA5-dependent IFN production, PKR activation, and cell death
The human RNA-editing enzyme ADAR1 prevents endogenous RNA from activating innate immune sensors (PKR, MDA5), which allows efficient translation during IFN response.
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.
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.
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.
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.
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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.
Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 ...presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.
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.
Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. HEV infections are the common cause of acute hepatitis but ...can also take chronic courses. Ribavirin is the treatment of choice for most patients and type I interferon (IFN) has been evaluated in a few infected transplantation patients in vivo. However, no effective and specific treatments against HEV infections are currently available.
In this study, we evaluated the natural compound silvestrol, isolated from the plant Aglaia foveolata, and known for its specific inhibition of the DEAD-box RNA helicase eIF4A in state-of-the-art HEV experimental model systems. Silvestrol blocked HEV replication of different subgenomic replicons in a dose-dependent manner at low nanomolar concentrations and acted additive to ribavirin (RBV). In addition, HEV p6-based full length replication and production of infectious particles was reduced in the presence of silvestrol. A pangenotypic effect of the compound was further demonstrated with primary isolates from four different human genotypes in HEV infection experiments of hepatocyte-like cells derived from human embryonic and induced pluripotent stem cells. In vivo, HEV RNA levels rapidly declined in the feces of treated mice while no effect was observed in the vehicle treated control animals. In conclusion, silvestrol could be identified as pangenotypic HEV replication inhibitor in vitro with additive effect to RBV and further demonstrated high potency in vivo. The compound therefore may be considered in future treatment strategies of chronic hepatitis E in immunocompromised patients.
•The natural compound silvestrol is a potent inhibitor of HEV replication.•HEV infection of laboratory and primary isolates could be inhibited by silvestrol.•Silvestrol demonstrated high potency in human liver chimeric mice.•Targeting translation initiation could be a novel antiviral strategy for the treatment of chronic hepatitis E.
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