Bat pluripotent stem cells reveal unusual entanglement between host and viruses.

Bats are distinctive among mammals due to their ability to fly, use laryngeal echolocation, and tolerate viruses. However, there are currently no reliable cellular models for studying bat biology or their response to viral infections. Here, we created induced pluripotent stem cells (iPSCs) from two species of bats: the wild greater horseshoe bat (Rhinolophus ferrumequinum) and the greater mouse-eared bat (Myotis myotis). The iPSCs from both bat species showed similar characteristics and had a gene expression profile resembling that of cells attacked by viruses. They also had a high number of endogenous viral sequences, particularly retroviruses. These results suggest that bats have evolved mechanisms to tolerate a large load of viral sequences and may have a more intertwined relationship with viruses than previously thought. Further study of bat iPSCs and their differentiated progeny will provide insights into bat biology, virus host relationships, and the molecular basis of bats' special traits.

Pluripotency and nuclear reprogramming.

Pluripotency is a "blank" cellular state characteristic of specific cells within the early embryo (e.g., epiblast cells) and of certain cells propagated in vitro (e.g., embryonic stem cells, ESCs). The terms pluripotent cell and stem cell are often used interchangeably to describe cells capable of differentiating into multiple cell types. In this review, we discuss the prevailing molecular and functional definitions of pluripotency and the working parameters employed to describe this state, both in the context of cells residing within the early embryo and cells propagated in vitro.

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.

Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development.

During implantation, the murine embryo transitions from a "quiet" into an active metabolic/proliferative state, which kick-starts the growth and morphogenesis of the post-implantation conceptus. Such transition is also required for embryonic stem cells to be established from mouse blastocysts, but the factors regulating this process are poorly understood. Here, we show that Ronin plays a critical role in the process by enabling active energy production, and the loss of Ronin results in the establishment of a reversible quiescent state in which naïve pluripotency is promoted. In addition, Ronin fine-tunes the expression of genes that encode ribosomal proteins and is required for proper tissue-scale organisation of the pluripotent lineage during the transition from blastocyst to egg cylinder stage. Thus, Ronin function is essential for governing the metabolic capacity so that it can support the pluripotent lineage's high-energy demands for cell proliferation and morphogenesis.

Ronin is essential for embryogenesis and the pluripotency of mouse embryonic stem cells.

Pluripotency is a unique biological state that allows cells to differentiate into any tissue type. Here we describe a candidate pluripotency factor, Ronin, that possesses a THAP domain, which is associated with sequence-specific DNA binding and epigenetic silencing of gene expression. Ronin is expressed primarily during the earliest stages of murine embryonic development, and its deficiency in mice produces periimplantational lethality and defects in the inner cell mass. Conditional knockout of Ronin prevents the growth of ES cells while forced expression of Ronin allows ES cells to proliferate without differentiation under conditions that normally do not promote self-renewal. Ectopic expression also partly compensates for the effects of Oct4 knockdown. We demonstrate that Ronin binds directly to HCF-1, a key transcriptional regulator. Our findings identify Ronin as an essential factor underlying embryogenesis and ES cell pluripotency. Its association with HCF-1 suggests an epigenetic mechanism of gene repression in pluripotent cells.

Ronin/Hcf-1 binds to a hyperconserved enhancer element and regulates genes involved in the growth of embryonic stem cells.

Self-renewing embryonic stem (ES) cells have an exceptional need for timely biomass production, yet the transcriptional control mechanisms responsible for meeting this requirement are largely unknown. We report here that Ronin (Thap11), which is essential for the self-renewal of ES cells, binds with its transcriptional coregulator, Hcf-1, to a highly conserved enhancer element that previously lacked a recognized binding factor. The subset of genes bound by Ronin/Hcf-1 function primarily in transcription initiation, mRNA splicing, and cell metabolism; genes involved in cell signaling and cell development are conspicuously underrepresented in this target gene repertoire. Although Ronin/Hcf-1 represses the expression of some target genes, its activity at promoter sites more often leads to the up-regulation of genes essential to protein biosynthesis and energy production. We propose that Ronin/Hcf-1 controls a genetic program that contributes to the unimpeded growth of ES cells.

Generation of human pluripotent stem cell lines (WAe009-A) with THAP11F80L cobalamin disorder-associated mutation.

Recent studies reported that the mutation in the THAP11 gene (THAP11F80L) could be responsible for the inborn vitamin deficiency known as cobalamin disorder, by affecting the expression of the enzyme MMACHC, key in the cobalamin metabolism. However, the specifics of the molecular mechanism are largely unknown. In here we generated genetically modified human pluripotent stem cell lines with THAP11F80L mutation, providing a new research tool for futher exploring the molecular mechanism. The established hPSC lines remain pluripotent, showing expression of OCT3/4, differentiation capacity to the three germ layers and displaying normal karyotype.

Regulatory architecture of housekeeping genes is driven by promoter assemblies.

Genes that are key to cell identity are generally regulated by cell-type-specific enhancer elements bound by transcription factors, some of which facilitate looping to distant gene promoters. In contrast, genes that encode housekeeping functions, whose regulation is essential for normal cell metabolism and growth, generally lack interactions with distal enhancers. We find that Ronin (Thap11) assembles multiple promoters of housekeeping and metabolic genes to regulate gene expression. This behavior is analogous to how enhancers are brought together with promoters to regulate cell identity genes. Thus, Ronin-dependent promoter assemblies provide a mechanism to explain why housekeeping genes can forgo distal enhancer elements and why Ronin is important for cellular metabolism and growth control. We propose that clustering of regulatory elements is a mechanism common to cell identity and housekeeping genes but is accomplished by different factors binding distinct control elements to establish enhancer-promoter or promoter-promoter interactions, respectively.

Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity.

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.

Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission.

SARS-CoV-2 lineages have diverged into highly prevalent variants termed "variants of concern" (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission.

The oral drug nitazoxanide restricts SARS-CoV-2 infection and attenuates disease pathogenesis in Syrian hamsters.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs.

Ronin overexpression induces cerebellar degeneration in a mouse model of ataxia.

Spinocerebellar ataxias (SCAs) are a group of genetically heterogeneous inherited neurodegenerative disorders characterized by progressive ataxia and cerebellar degeneration. Here, we used a mouse model to test a possible connection between SCA and Ronin (Thap11), a polyglutamine-containing transcriptional regulator encoded in a region of human chromosome 16q22.1 that has been genetically linked to SCA type 4. We report that transgenic expression of Ronin in mouse cerebellar Purkinje cells leads to detrimental loss of these cells and the development of severe ataxia as early as 10 weeks after birth. Mechanistically, we find that several SCA-causing genes harbor Ronin DNA-binding motifs and are transcriptionally deregulated in transgenic animals. In addition, ectopic expression of Ronin in embryonic stem cells significantly increases the protein level of Ataxin-1, the protein encoded by Atxn1, alterations of which cause SCA type 1. This increase is also seen in the cerebellum of transgenic animals, although the latter was not statistically significant. Hence, our data provide evidence for a link between Ronin and SCAs, and suggest that Ronin may be involved in the development of other neurodegenerative diseases.

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

Immature mDA neurons ameliorate motor deficits in a 6-OHDA Parkinson's disease mouse model and are functional after cryopreservation.

Parkinson's disease is associated with the loss of dopaminergic neurons in the midbrain. Clinical studies investigating replacement of these neurons with in vitro-generated neurons are currently underway. However, this approach has been limited by difficulties in scaling up on-demand production of midbrain dopaminergic (mDA) neurons from pluripotent stem cells. Cryo-preservation may offer a solution, as it allows for banking of quality controlled mDA neurons. In this study, we tested different freezing conditions and found that optimal cryopreservation of immature human mDA neurons at an early differentiation time point was achieved in STEM-CELLBANKER medium using a controlled freezing program.

FACS-Mediated Isolation of Neuronal Cell Populations From Virus-Infected Human Embryonic Stem Cell-Derived Cerebral Organoid Cultures.

Organoids-or pluripotent stem cell-derived in vitro-grown simplified mini organs-have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence-assisted cell sorting (FACS) of virus-infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type-specific multi-omics analyses. The protocol starts from stem cell-derived mature brain organoids and includes steps for: preparing the culture for viral infection, production of the viral stocks, FACS sample preparation, and gating and sorting implementation. The protocol has been developed for Zika virus infection, but can be extrapolated to other viruses or fluorescent marker expression as illustrated in an alternate protocol using a single-cycle lentivirus expressing a fluorescent reporter protein. © 2018 by John Wiley & Sons, Inc.

Generation of hiPSTZ16 (ISMMSi003-A) cell line from normal human foreskin fibroblasts.

Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A). These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source- and reprogramming method-independent effects in downstream applications.

Zika Virus Alters DNA Methylation of Neural Genes in an Organoid Model of the Developing Human Brain.

Zika virus (ZIKV) infection during early pregnancy can cause microcephaly and associated defects at birth, but whether it can induce neurologic sequelae that appear later in life remains unclear. Using a model of the developing brain based on embryonic stem cell-derived brain organoids, we studied the impact of ZIKV infection on the DNA methylation pattern across the entire genome in selected neural cell types. The virus unexpectedly alters the DNA methylome of neural progenitors, astrocytes, and differentiated neurons at genes that have been implicated in the pathogenesis of a number of brain disorders, most prominently mental retardation and schizophrenia. Our results suggest that ZIKV infection during fetal development could lead to a spectrum of delayed-onset neuropsychiatric complications. IMPORTANCE Scientific research on human neural stem cells and cerebral organoids has confirmed the congenital neurotropic and neurodestructive nature of the Zika virus. However, the extent to which prenatal ZIKV infection is associated with more subtle brain alterations, such as epigenetic changes, remains ill defined. Here, we address the question of whether ZIKV infection induces DNA methylation changes with the potential to cause brain disorders later in life.

Ronin influences the DNA damage response in pluripotent stem cells.

Early mammalian embryonic cells must maintain a particularly robust DNA repair system, as mutations at this developmental point have detrimental consequences for the organism. How the repair system can be tuned to fulfill such elevated requirements is largely unknown, but it may involve transcriptional regulation. Ronin (Thap11) is a transcriptional regulator responsible for vital programs in pluripotent cells. Here, we report that this protein also modulates the DNA damage response of such cells. We show that conditional Ronin knockout sensitizes embryonic stem cells (ESCs) to UV-C-induced DNA damage in association with Atr pathway activation and G2/M arrest. Ronin binds to and regulates the genes encoding several DNA repair factors, including Gtf2h4 and Rad18, providing a potential mechanism for this phenotype. Our results suggest that the unique DNA repair requirements of the early embryo are not met by a static system, but rather via highly regulated processes.

Ronin Governs Early Heart Development by Controlling Core Gene Expression Programs.

Ronin (THAP11), a DNA-binding protein that evolved from a primordial DNA transposon by molecular domestication, recognizes a hyperconserved promoter sequence to control developmentally and metabolically essential genes in pluripotent stem cells. However, it remains unclear whether Ronin or related THAP proteins perform similar functions in development. Here, we present evidence that Ronin functions within the nascent heart as it arises from the mesoderm and forms a four-chambered organ. We show that Ronin is vital for cardiogenesis during midgestation by controlling a set of critical genes. The activity of Ronin coincided with the recruitment of its cofactor, Hcf-1, and the elevation of H3K4me3 levels at specific target genes, suggesting the involvement of an epigenetic mechanism. On the strength of these findings, we propose that Ronin activity during cardiogenesis offers a template to understand how important gene programs are sustained across different cell types within a developing organ such as the heart.