Western Zika Virus in Human Fetal Neural Progenitors Persists Long Term with Partial Cytopathic and Limited Immunogenic Effects

SummaryThe recent Zika virus (ZIKV) outbreak in the Western hemisphere is associated with severe pathology in newborns, including microcephaly and brain damage. The mechanisms underlying these outcomes are under intense investigation. Here, we show that a 2015 ZIKV isolate replicates in multiple cell types, including primary human fetal neural progenitors (hNPs). In immortalized cells, ZIKV is cytopathic and grossly rearranges endoplasmic reticulum membranes similar to other flaviviruses. In hNPs, ZIKV infection has a partial cytopathic phase characterized by cell rounding, pyknosis, and activation of caspase 3. Despite notable cell death, ZIKV did not activate a cytokine response in hNPs. This lack of cell intrinsic immunity to ZIKV is consistent with our observation that virus replication persists in hNPs for at least 28 days. These findings, supported by published fetal neuropathology, establish a proof-of-concept that neural progenitors in the developing human fetus can be direct targets of detrimental ZIKV-induced pathology

Dynamic Modulation of Thymic MicroRNAs in Response to Stress

thymocyte subsets. Several of the differentially regulated murine thymic miRs are also stress responsive in the heart, kidney, liver, brain, and/or spleen. The most dramatic thymic microRNA down modulated is miR-181d, exhibiting a 15-fold reduction following stress. This miR has both similar and distinct gene targets as miR-181a, another member of miR-181 family. Many of the differentially regulated microRNAs have known functions in thymopoiesis, indicating that their dysregulation will alter T cell repertoire selection and the formation of naïve T cells. This data has implications for clinical treatments involving anti-inflammatory steroids, ablation therapies, and provides mechanistic insights into the consequences of infections

Cell-Based Screen Identifies Human Interferon-Stimulated Regulators of <i>Listeria monocytogenes</i> Infection

<div><p>The type I interferon (IFN) activated transcriptional response is a critical antiviral defense mechanism, yet its role in bacterial pathogenesis remains less well characterized. Using an intracellular pathogen <i>Listeria monocytogenes</i> (<i>Lm</i>) as a model bacterial pathogen, we sought to identify the roles of individual interferon-stimulated genes (ISGs) in context of bacterial infection. Previously, IFN has been implicated in both restricting and promoting <i>Lm</i> growth and immune stimulatory functions <i>in vivo</i>. Here we adapted a gain-of-function flow cytometry based approach to screen a library of more than 350 human ISGs for inhibitors and enhancers of <i>Lm</i> infection. We identify 6 genes, including <i>UNC93B1</i>, <i>MYD88</i>, <i>AQP9</i>, and <i>TRIM14</i> that potently inhibit <i>Lm</i> infection. These inhibitors act through both transcription-mediated (MYD88) and non-transcriptional mechanisms (TRIM14). Further, we identify and characterize the human high affinity immunoglobulin receptor FcγRIa as an enhancer of <i>Lm</i> internalization. Our results reveal that FcγRIa promotes <i>Lm</i> uptake in the absence of known host <i>Lm</i> internalization receptors (E-cadherin and c-Met) as well as bacterial surface internalins (InlA and InlB). Additionally, FcγRIa-mediated uptake occurs independently of <i>Lm</i> opsonization or canonical FcγRIa signaling. Finally, we established the contribution of FcγRIa to <i>Lm</i> infection in phagocytic cells, thus potentially linking the IFN response to a novel bacterial uptake pathway. Together, these studies provide an experimental and conceptual basis for deciphering the role of IFN in bacterial defense and virulence at single-gene resolution.</p></div

The mechanism of RNA capping by SARS-CoV-2

The RNA genome of SARS-CoV-2 contains a 5′ cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1–4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2′-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2′-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA–protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication–transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19

The CD3 zeta subunit contains a phosphoinositide-binding motif that is required for the stable accumulation of TCR-CD3 complex at the immunological synapse

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. While all the CD3 subunits possess at least one ITAM, CD3 ε subunit also contains a proline-rich sequence (PRS) and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. Herein, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to stably accumulate at the immunological synapse during T cell-antigen presenting cell interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures following T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation

Developing a cellular model of FcγRIa function.

<p>(A) Diagram illustrating the phagocytic assay used to reconstitute FcγR function using Alexa Fluor 488 IgG (green) and DyLight 405 (blue) IgG-labeled polystyrene beads. (B) Representative fluorescence microscopy images of U-2 OS cells transduced with lentivirus co-expressing TagRFP and Fluc, FcγRIa, or FcγRIIa, incubated with Alexa Fluor 488 IgG-opsonized beads (green) for 1.5 h, followed by secondary DyLight 405 IgG labeling (blue) of external beads. (C) Quantification of phagocytosed IgG-coated beads. Error bars represent s.d., 160 cells were counted for each of three independent experiments.</p

FcγRIa-mediated <i>Lm</i> invasion exhibits host species tropism.

<p>(A) Representative fluorescence microscopy images of U-2 OS cells transduced with lentivirus co-expressing TagRFP and Fluc or FcγRIa from indicated species incubated with Alexa Fluor 488 IgG-opsonized beads (green) for 1.5 h. (B) Quantification of phagocytosed human IgG-coated beads in U-2 OS cells transduced with lentivirus expressing γ-chain and FcγRIa of indicated species. Error bars represent s.d., 40 cells were counted for each of the four independent experiments (n.s., not significant). (C) Infectivity of <i>Lm ΔinlAΔinlB</i> in HEK293A cells transduced with lentivirus expressing Fluc (white bar), human FcγRIa (black bar) or FcγRIa from indicated species (grey bars). <i>Lm</i> infectivity was measured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006102#ppat.1006102.g002" target="_blank">Fig 2C</a>, n = 3, error bars represent s.d. (****, P<0.0001). (D) Infectivity of wild type <i>Lm</i> in MEFs transduced with lentivirus expressing human γ-chain and Fluc or murine FcγRIa. <i>Lm</i> infectivity was measured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006102#ppat.1006102.g002" target="_blank">Fig 2C</a>, error bars represent s.d., n = 3, statistical significance was determined by t-test prior to normalization (n.s., not significant).</p

Flow cytometry-based gain-of-function screen identifies regulators of <i>Lm</i> infection.

<p>(A) Schematic of the bicistronic lentiviral vector; CMV, immediate early promoter from human cytomegalovirus; LTR, HIV-1 long terminal repeat. (B) Diagram illustrating the gain-of-function fluorescence-based screen for regulators of <i>Lm</i> infection. (C) YFV and <i>Lm</i> infectivity in the presence of ISG inhibitors and enhancers of viral infection. Infectivity was measured by flow cytometry as a percentage of GFP-positive cells in RFP-positive population and normalized to a Fluc control for each pathogen (white bars). Error bars represent s.d., n = 3 (YFV), n = 2 (<i>Lm</i>). Statistical significance was determined by one-way analysis of variance (ANOVA) for each pathogen prior to normalization (****, P<0.0001; n.s., not significant). (D) (<i>Left</i>) Dot plot of <i>Lm</i> infectivity in the presence of expressed ISGs. <i>Lm</i> infectivity was measured by flow cytometry in two replicate screens and presented as an average. Error bars represent s.d., n = 2. (<i>Right</i>) Scatter plot of Z-scores of screen replicates 1 and 2. Genes selected for further confirmation are labeled (<i>left</i>) and boxed (<i>right</i>). (E) Infectivity of <i>Lm</i> in <i>STAT1</i>-deficient fibroblasts transduced with lentivirus expressing Fluc (white bar) and selected ISGs from the large-scale screen in (D). <i>Lm</i> infectivity was measured similarly to Fig 2C. Error bars represent s.d., n = 3 (*, P<0.05; ****, P<0.0001; n.s., not significant).</p