Invading pathogen nucleic acids are recognized and bound by cytoplasmic (retinoic acid-inducible gene I RIG-I-like) and membrane-bound (Toll-like) pattern recognition receptors to activate innate ...immune signaling. Modified nucleotides, when present in RNA molecules, diminish the magnitude of these signaling responses. However, mechanisms explaining the blunted signaling have not been elucidated. In this study, we used several independent biological assays, including inhibition of virus replication, RIG-I:RNA binding assays, and limited trypsin digestion of RIG-I:RNA complexes, to begin to understand how RNAs containing modified nucleotides avoid or suppress innate immune signaling. The experiments were based on a model innate immune activating RNA molecule, the polyU/UC RNA domain of hepatitis C virus, which was transcribed in vitro with canonical nucleotides or with one of eight modified nucleotides. The approach revealed signature assay responses associated with individual modified nucleotides or classes of modified nucleotides. For example, while both N-6-methyladenosine (m6A) and pseudouridine nucleotides correlate with diminished signaling, RNA containing m6A modifications bound RIG-I poorly, while RNA containing pseudouridine bound RIG-I with high affinity but failed to trigger the canonical RIG-I conformational changes associated with robust signaling. These data advance understanding of RNA-mediated innate immune signaling, with additional relevance for applying nucleotide modifications to RNA therapeutics.
The innate immune system provides the first response to virus infections and must distinguish between host and pathogen nucleic acids to mount a protective immune response without activating autoimmune responses. While the presence of nucleotide modifications in RNA is known to correlate with diminished innate immune signaling, the underlying mechanisms have not been explored. The data reported here are important for defining mechanistic details to explain signaling suppression by RNAs containing modified nucleotides. The results suggest that RNAs containing modified nucleotides interrupt signaling at early steps of the RIG-I-like innate immune activation pathway and also that nucleotide modifications with similar chemical structures can be organized into classes that suppress or evade innate immune signaling steps. These data contribute to defining the molecular basis for innate immune signaling suppression by RNAs containing modified nucleotides. The results have important implications for designing therapeutic RNAs that evade innate immune detection.
The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid ...design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises.
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•A portable, low-cost diagnostic platform for the detection of Zika virus•Discrimination of viral strains at single-base resolution using a CRISPR-based tool•Low femtomolar detection of Zika virus from infected monkey plasma•Programmable sensor development workflow for rapid responses to global epidemics
A diagnostic platform utilizing biomolecular sensors and CRISPR-based technology allows rapid, specific, and low-cost detection of the Zika virus at clinically relevant concentrations.
An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural ...progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry, expansion of the progenitor population, and delayed neuronal differentiation, all key features of the developing human cortex. In contrast, Pten deletion in mouse organoids does not lead to folding. Finally, we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex.
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•PTEN deletion enhances human NP proliferation and sensitivity to growth factors•PTEN mutant human, but not mouse, cerebral organoids display surface folding•Folded human organoids contain expanded ventricular and outer progenitors•Zika virus impairs the formation of expanded and folded human cerebral organoids
The growth and structural formation of the human cortex can be modeled in vitro using cerebral organoids. In this article, Li, Muffat, and colleagues show that enhancing the PTEN-AKT signaling pathway leads to the generation of human cerebral organoids that are expanded in size and display surface folding.
Maternal Zika virus (ZIKV) infection during pregnancy is recognized as the cause of an epidemic of microcephaly and other neurological anomalies in human fetuses. It remains unclear how ZIKV accesses ...the highly vulnerable population of neural progenitors of the fetal central nervous system (CNS), and which cell types of the CNS may be viral reservoirs. In contrast, the related dengue virus (DENV) does not elicit teratogenicity. To model viral interaction with cells of the fetal CNS in vitro, we investigated the tropism of ZIKV and DENV for different induced pluripotent stem cell-derived human cells, with a particular focus on microglia-like cells. We show that ZIKV infected isogenic neural progenitors, astrocytes, and microglia-like cells (pMGLs), but was only cytotoxic to neural progenitors. Infected glial cells propagated ZIKV and maintained ZIKV load over time, leading to viral spread to susceptible cells. DENV triggered stronger immune responses and could be cleared by neural and glial cells more efficiently. pMGLs, when cocultured with neural spheroids, invaded the tissue and, when infected with ZIKV, initiated neural infection. Since microglia derive from primitive macrophages originating in proximity to the maternal vasculature, they may act as a viral reservoir for ZIKV and establish infection of the fetal brain. Infection of immature neural stem cells by invading microglia may occur in the early stages of pregnancy, before angiogenesis in the brain rudiments. Our data are also consistent with ZIKV and DENV affecting the integrity of the blood–brain barrier, thus allowing infection of the brain later in life.
Field-deployable viral diagnostics using CRISPR-Cas13 Myhrvold, Cameron; Freije, Catherine A; Gootenberg, Jonathan S ...
Science (American Association for the Advancement of Science),
04/2018, Volume:
360, Issue:
6387
Journal Article
Peer reviewed
Open access
Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific ...high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015-2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms.
Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of ...type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.
Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV ...infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention.
Brain organoids grown from human pluripotent stem cells self-organize into cytoarchitectures resembling the developing human brain. These three-dimensional models offer an unprecedented opportunity ...to study human brain development and dysfunction. Characterization currently sacrifices spatial information for single-cell or histological analysis leaving whole-tissue analysis mostly unexplored. Here, we present the SCOUT pipeline for automated multiscale comparative analysis of intact cerebral organoids. Our integrated technology platform can rapidly clear, label, and image intact organoids. Algorithmic- and convolutional neural network-based image analysis extract hundreds of features characterizing molecular, cellular, spatial, cytoarchitectural, and organoid-wide properties from fluorescence microscopy datasets. Comprehensive analysis of 46 intact organoids and ~ 100 million cells reveals quantitative multiscale "phenotypes" for organoid development, culture protocols and Zika virus infection. SCOUT provides a much-needed framework for comparative analysis of emerging 3D in vitro models using fluorescence microscopy.
Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia
. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching ...through variation in its expression during development
. 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.
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