Targeting cellular cathepsins inhibits hepatitis E virus entry.

BACKGROUND AND AIMS: HEV is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potential of cellular protease during HEV infection. APPROACH AND RESULTS: Using our established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 suppressed HEV infections with an EC 50 of ~0.02 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells, was also observed in HepaRG and primary human hepatocytes. Furthermore, through time-of-addition and RNAscope experiments, we confirmed that HEV entry is blocked by inhibition of cathepsins. Cathepsin L (CTSL) knockout cells were less permissive to HEV, suggesting that CTSL is critical for HEV infection. Finally, we observed cleavage of the glycosylated ORF2 protein and virus particles by recombinant CTSL. CONCLUSIONS: In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor K11777, especially with its notable safety profile in primary cells, further underscores its potential as a therapeutic candidate.

Single-cell brain organoid screening identifies developmental defects in autism.

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1-4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR-human organoids-single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR-Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.

Low-density lipoprotein receptor-related protein 1 (LRP1) as an auxiliary host factor for RNA viruses.

Viruses with an RNA genome are often the cause of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that are resistant to Rift Valley fever virus (RVFV). This screen returned the low-density lipoprotein receptor-related protein 1 (LRP1) as a top hit, a plasma membrane protein involved in a wide variety of cell activities. Inactivation of LRP1 in human cells reduced RVFV RNA levels already at the attachment and entry stages of infection. Moreover, the role of LRP1 in promoting RVFV infection was dependent on physiological levels of cholesterol and on endocytosis. In the human cell line HuH-7, LRP1 also promoted early infection stages of sandfly fever Sicilian virus and La Crosse virus, but had a minor effect on late infection by vesicular stomatitis virus, whereas encephalomyocarditis virus was entirely LRP1-independent. Moreover, siRNA experiments in human Calu-3 cells demonstrated that also SARS-CoV-2 infection benefitted from LRP1. Thus, we identified LRP1 as a host factor that supports infection by a spectrum of RNA viruses.

EGF receptor modulates HEV entry in human hepatocytes.

BACKGROUND AND AIMS: Being the most common cause of acute viral hepatitis with >20 million cases per year and 70,000 deaths annually, HEV presents a long-neglected and underinvestigated health burden. Although the entry process of viral particles is an attractive target for pharmacological intervention, druggable host factors to restrict HEV entry have not been identified so far. APPROACH AND RESULTS: Here we identify the EGF receptor (EGFR) as a novel host factor for HEV and reveal the significance of EGFR for the HEV entry process. By utilizing RNAi, chemical modulation with Food and Drug Administration-approved drugs, and ectopic expression of EGFR, we revealed that EGFR is critical for HEV infection without affecting HEV RNA replication or assembly of progeny virus. We further unveiled that EGFR itself and its ligand-binding domain, rather than its signaling function, is responsible for the proviral effect. Modulation of EGF expression in HepaRG cells and primary human hepatocytes affected HEV infection. CONCLUSIONS: Taken together, our study provides novel insights into the life cycle of HEV and identified EGFR as a possible target for future antiviral strategies against HEV.

Hepatitis E virus is effectively inactivated by methylene blue plus light treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment with methylene blue (MB) and visible light is a well-established pathogen inactivation system for human plasma. This technique is routinely used in different countries. MB/light treatment was shown to inactivate several transfusion-transmittable viruses, but its efficiency for the inactivation of the quasi-enveloped hepatitis E virus (HEV) has not yet been investigated. MATERIALS AND METHODS: Plasma units were spiked with cell culture-derived HEV and treated with the THERAFLEX MB-Plasma system using various light doses (30, 60, 90, and 120 J/cm2 ). HEV titers in pre- and post-treatment samples were determined by virus titration and a large-volume plating assay to improve the detection limit of the virus assay. RESULTS: THERAFLEX MB-Plasma efficiently inactivated HEV in human plasma. Even the lowest light dose of 30 J/cm2 inactivated HEV down to the limit of detection, with a mean log reduction factor of greater than 2.4 for the total process. CONCLUSION: Our study demonstrates that the THERAFLEX MB-Plasma system effectively inactivates HEV in human plasma.

Viral Interference of Hepatitis C and E Virus Replication in Novel Experimental Co-Infection Systems.

BACKGROUND: Hepatitis C virus (HCV) constitutes a global health problem, while hepatitis E virus (HEV) is the major cause of acute viral hepatitis globally. HCV/HEV co-infections have been poorly characterized, as they are hampered by the lack of robust HEV cell culture systems. This study developed experimental models to study HCV/HEV co-infections and investigate viral interference in cells and humanized mice. METHODS: We used state-of-the art human hepatocytes tissue culture models to assess HEV and HCV replication in co- or super-transfection settings. Findings were confirmed by co- and super-infection experiments in human hepatocytes and in vivo in human liver chimeric mice. RESULTS: HEV was inhibited by concurrent HCV replication in human hepatocytes. This exclusion phenotype was linked to the protease activity of HCV. These findings were corroborated by the fact that in HEV on HCV super-infected mice, HEV viral loads were reduced in individual mice. Similarly, HCV on HEV super-infected mice showed reduced HCV viral loads. CONCLUSION: Direct interference of both viruses with HCV NS3/4A as the determinant was observed. In vivo, we detected reduced replication of both viruses after super-infection in individual mice. These findings provide new insights into the pathogenesis of HCV-HEV co-infections and should contribute to its clinical management in the future.

Systematic refinement of gene annotations by parsing mRNA 3' end sequencing datasets.

Alternative cleavage and polyadenylation generates mRNA 3' isoforms in a cell type-specific manner. Due to finite available RNA sequencing data of organisms with vast cell type complexity, currently available gene annotation resources are incomplete, which poses significant challenges to the comprehensive interpretation and quantification of transcriptomes. In this chapter, we introduce 3'GAmES, a stand-alone computational pipeline for the identification and quantification of novel mRNA 3'end isoforms from 3'mRNA sequencing data. 3'GAmES expands available repositories and improves comprehensive gene-tag counting by cost-effective 3' mRNA sequencing, faithfully mirroring whole-transcriptome RNAseq measurements. By employing R and bash shell scripts (assembled in a Singularity container) 3'GAmES systematically augments cell type-specific 3' ends of RNA polymerase II transcripts and increases the sensitivity of quantitative gene expression profiling by 3' mRNA sequencing. Public access: https://github.com/AmeresLab/3-GAmES.git.

Rapid nucleus-scale reorganization of chromatin in neurons enables transcriptional adaptation for memory consolidation.

The interphase nucleus is functionally organized in active and repressed territories defining the transcriptional status of the cell. However, it remains poorly understood how the nuclear architecture of neurons adapts in response to behaviorally relevant stimuli that trigger fast alterations in gene expression patterns. Imaging of fluorescently tagged nucleosomes revealed that pharmacological manipulation of neuronal activity in vitro and auditory cued fear conditioning in vivo induce nucleus-scale restructuring of chromatin within minutes. Furthermore, the acquisition of auditory fear memory is impaired after infusion of a drug into auditory cortex which blocks chromatin reorganization in vitro. We propose that active chromatin movements at the nucleus scale act together with local gene-specific modifications to enable transcriptional adaptations at fast time scales. Introducing a transgenic mouse line for photolabeling of histones, we extend the realm of systems available for imaging of chromatin dynamics to living animals.

Organoid modeling of Zika and herpes simplex virus 1 infections reveals virus-specific responses leading to microcephaly.

Viral infection in early pregnancy is a major cause of microcephaly. However, how distinct viruses impair human brain development remains poorly understood. Here we use human brain organoids to study the mechanisms underlying microcephaly caused by Zika virus (ZIKV) and herpes simplex virus (HSV-1). We find that both viruses efficiently replicate in brain organoids and attenuate their growth by causing cell death. However, transcriptional profiling reveals that ZIKV and HSV-1 elicit distinct cellular responses and that HSV-1 uniquely impairs neuroepithelial identity. Furthermore, we demonstrate that, although both viruses fail to potently induce the type I interferon system, the organoid defects caused by their infection can be rescued by distinct type I interferons. These phenotypes are not seen in 2D cultures, highlighting the superiority of brain organoids in modeling viral infections. These results uncover virus-specific mechanisms and complex cellular immune defenses associated with virus-induced microcephaly.

Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters.

We developed a functional lineage tracing tool termed CaTCH (CRISPRa tracing of clones in heterogeneous cell populations). CaTCH combines precise clonal tracing of millions of cells with the ability to retrospectively isolate founding clones alive before and during selection, allowing functional experiments. Using CaTCH, we captured rare clones representing as little as 0.001% of a population and investigated the emergence of resistance to targeted melanoma therapy in vivo.

Structure-function analysis of microRNA 3'-end trimming by Nibbler.

Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/ß-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn2+-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.

Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles.

CRISPR-Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a 'Bioscore' and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR-Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.

Robust hepatitis E virus infection and transcriptional response in human hepatocytes.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.

DigiTAG-a RNA Sequencing Approach to Analyze Transcriptomes of Rare Cell Populations in Drosophila melanogaster.

Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. Here we describe DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.

Dynamics of activating and repressive histone modifications in Drosophila neural stem cell lineages and brain tumors.

During central nervous system development, spatiotemporal gene expression programs mediate specific lineage decisions to generate neuronal and glial cell types from neural stem cells (NSCs). However, little is known about the epigenetic landscape underlying these highly complex developmental events. Here, we perform ChIP-seq on distinct subtypes of Drosophila FACS-purified NSCs and their differentiated progeny to dissect the epigenetic changes accompanying the major lineage decisions in vivo By analyzing active and repressive histone modifications, we show that stem cell identity genes are silenced during differentiation by loss of their activating marks and not via repressive histone modifications. Our analysis also uncovers a new set of genes specifically required for altering lineage patterns in type II neuroblasts (NBs), one of the two main Drosophila NSC identities. Finally, we demonstrate that this subtype specification in NBs, unlike NSC differentiation, requires Polycomb-group-mediated repression.

The transcription factor odd-paired regulates temporal identity in transit-amplifying neural progenitors via an incoherent feed-forward loop.

Neural progenitors undergo temporal patterning to generate diverse neurons in a chronological order. This process is well-studied in the developing Drosophila brain and conserved in mammals. During larval stages, intermediate neural progenitors (INPs) serially express Dichaete (D), grainyhead (Grh) and eyeless (Ey/Pax6), but how the transitions are regulated is not precisely understood. Here, we developed a method to isolate transcriptomes of INPs in their distinct temporal states to identify a complete set of temporal patterning factors. Our analysis identifies odd-paired (opa), as a key regulator of temporal patterning. Temporal patterning is initiated when the SWI/SNF complex component Osa induces D and its repressor Opa at the same time but with distinct kinetics. Then, high Opa levels repress D to allow Grh transcription and progress to the next temporal state. We propose that Osa and its target genes opa and D form an incoherent feedforward loop (FFL) and a new mechanism allowing the successive expression of temporal identities.

Estrogen Signaling Drives Ciliogenesis in Human Endometrial Organoids.

The human endometrium is the inner lining of the uterus consisting of stromal and epithelial (secretory and ciliated) cells. It undergoes a hormonally regulated monthly cycle of growth, differentiation, and desquamation. However, how these cyclic changes control the balance between secretory and ciliated cells remains unclear. Here, we established endometrial organoids to investigate the estrogen (E2)-driven control of cell fate decisions in human endometrial epithelium. We demonstrate that they preserve the structure, expression patterns, secretory properties, and E2 responsiveness of their tissue of origin. Next, we show that the induction of ciliated cells is orchestrated by the coordinated action of E2 and NOTCH signaling. Although E2 is the primary driver, inhibition of NOTCH signaling provides a permissive environment. However, inhibition of NOTCH alone is not sufficient to trigger ciliogenesis. Overall, we provide insights into endometrial biology and propose endometrial organoids as a robust and powerful model for studying ciliogenesis in vitro.

Hepatitis E Virus Drug Development.

Hepatitis E virus (HEV) is an underestimated disease, leading to estimated 20 million infections and up to 70,000 deaths annually. Infections are mostly asymptomatic but can reach mortality rates up to 25% in pregnant women or become chronic in immunocompromised patients. The current therapy options are limited to the unspecific antivirals Ribavirin (RBV) and pegylated Interferon-α (pegIFN-α). RBV leads to viral clearance in only 80% of patients treated, and is, similar to pegIFN-α, contraindicated in the major risk group of pregnant women, emphasizing the importance of new therapy options. In this review, we focus on the urgent need and current efforts in HEV drug development. We provide an overview of the current status of HEV antiviral research. Furthermore, we discuss strategies for drug development and the limitations of the approaches with respect to HEV.

Publisher Correction: Guided self-organization and cortical plate formation in human brain organoids.

This corrects the article DOI: 10.1038/nbt.3906.