Zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning

Zika virus (ZIKV) causes congenital microcephaly. Although ZIKV can impair cell cycle progression and provoke apoptosis, which probably contributes to disease aetiology through depletion of neural progenitor cells, additional cellular mechanisms may be important. Here, we investigated whether ZIKV infection alters centrosome number and spindle positioning, because such defects are thought to be at the root of inherited primary autosomal recessive microcephaly (MCPH). In addition to HeLa cells, in which centrosome number and spindle positioning can be well monitored, we analysed retinal epithelial cells (RPE-1), as well as brain-derived microglial (CHME-5) and neural progenitor (ReN) cells, using immunofluorescence. We established that ZIKV infection leads to supernumerary foci containing centriolar proteins that in some cases drive multipolar spindle assembly, as well as spindle positioning defects in HeLa, RPE-1 and CHME-5 cells, but not in ReN cells. We uncovered similar phenotypes in HeLa cells upon infection with dengue virus (DENV-2), another flavivirus that does not target brain cells and does not cause microcephaly. We conclude that infection with Flaviviridae can increase centrosome numbers and impair spindle positioning, thus potentially contributing to microcephaly in the case of Zika.

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Zika virus is transmitted in neural progenitor cells via cell-to-cell spread and infection is inhibited by the autophagy inducer trehalose

Journal of Virology ◽

10.1128/jvi.02024-20 ◽

2020 ◽

pp. JVI.02024-20

Author(s):

Alex E Clark ◽

Zhe Zhu ◽

Florian Krach ◽

Jeremy N Rich ◽

Gene W. Yeo ◽

...

Keyword(s):

Viral Replication ◽

Progenitor Cells ◽

Zika Virus ◽

Neural Progenitor Cells ◽

Neutralizing Antibody ◽

Neural Progenitor ◽

Health Concern ◽

Free Virus ◽

Zikv Infection ◽

Infected Cells

Zika virus (ZIKV) is a mosquito-borne human pathogen that causes congenital Zika syndrome and neurological symptoms in some adults. There are currently no approved treatments or vaccines for ZIKV, and exploration of therapies targeting host processes could avoid viral development of drug resistance. The purpose of our study was to determine if the non-toxic and widely used disaccharide trehalose, which showed antiviral activity against Human Cytomegalovirus (HCMV) in our previous work, could restrict ZIKV infection in clinically relevant neural progenitor cells (NPCs). Trehalose is known to induce autophagy, the degradation and recycling of cellular components. Whether autophagy is proviral or antiviral for ZIKV is controversial and depends on cell type and specific conditions used to activate or inhibit autophagy. We show here that trehalose treatment of NPCs infected with recent ZIKV isolates from Panama and Puerto Rico significantly reduces viral replication and spread. In addition, we demonstrate that ZIKV infection in NPCs spreads primarily cell-to-cell as an expanding infectious center, and NPCs are infected via contact with infected cells far more efficiently than by cell-free virus. Importantly, ZIKV was able to spread in NPCs in the presence of neutralizing antibody.Importance Zika virus causes birth defects and can lead to neurological disease in adults. While infection rates are currently low, ZIKV remains a public health concern with no treatment or vaccine available. Targeting a cellular pathway to inhibit viral replication is a potential treatment strategy that avoids development of antiviral resistance. We demonstrate in this study that the non-toxic autophagy-inducing disaccharide trehalose reduces spread and output of ZIKV in infected neural progenitor cells (NPCs), the major cells infected in the fetus. We show that ZIKV spreads cell-to-cell in NPCs as an infectious center and that NPCs are more permissive to infection by contact with infected cells than by cell-free virus. We find that neutralizing antibody does not prevent the spread of the infection in NPCs. These results are significant in demonstrating anti-ZIKV activity of trehalose and in clarifying the primary means of Zika virus spread in clinically relevant target cells.

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Natural variation in gene expression and Zika virus susceptibility revealed by villages of neural progenitor cells

10.1101/2021.11.08.467815 ◽

2021 ◽

Author(s):

Michael F Wells ◽

James Nemesh ◽

Sulagna Ghosh ◽

Jana M Mitchell ◽

Curtis J Mello ◽

...

Keyword(s):

Genetic Variation ◽

Progenitor Cells ◽

Zika Virus ◽

Neural Progenitor Cells ◽

Neural Progenitor ◽

Virus Infectivity ◽

Cellular Mechanisms ◽

Natural Genetic Variation ◽

The Impact

Variation in the human genome contributes to abundant diversity in human traits and vulnerabilities, but the underlying molecular and cellular mechanisms are not yet known, and will need scalable approaches to accelerate their recognition. Here, we advanced and applied an experimental platform that analyzes genetic, molecular, and phenotypic heterogeneity across cells from very many human donors cultured in a single, shared in vitro environment, with algorithms (Dropulation and Census-seq) for assigning phenotypes to individual donors. We used natural genetic variation and synthetic (CRISPR-Cas9) genetic perturbations to analyze the vulnerability of neural progenitor cells to infection with Zika virus. These analyses identified a common variant in the antiviral IFITM3 gene that regulated IFITM3 expression and explained most inter-individual variation in NPCs' susceptibility to Zika virus infectivity. These and other approaches could provide scalable ways to recognize the impact of genes and genetic variation on cellular phenotypes.

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Comparative Microarray Analysis of Proliferating and Differentiating Murine ENS Progenitor Cells

Stem Cells International ◽

10.1155/2016/9695827 ◽

2016 ◽

Vol 2016 ◽

pp. 1-13 ◽

Cited By ~ 5

Author(s):

Peter Helmut Neckel ◽

Roland Mohr ◽

Ying Zhang ◽

Bernhard Hirt ◽

Lothar Just

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Neural Progenitor Cells ◽

Neural Progenitor ◽

Developmental Dysplasia ◽

Cellular Mechanisms ◽

Differentiation Markers ◽

Early Differentiation

Postnatal neural progenitor cells of the enteric nervous system are a potential source for future cell replacement therapies of developmental dysplasia like Hirschsprung’s disease. However, little is known about the molecular mechanisms driving the homeostasis and differentiation of this cell pool. In this work, we conducted Affymetrix GeneChip experiments to identify differences in gene regulation between proliferation and early differentiation of enteric neural progenitors from neonatal mice. We detected a total of 1333 regulated genes that were linked to different groups of cellular mechanisms involved in cell cycle, apoptosis, neural proliferation, and differentiation. As expected, we found an augmented inhibition in the gene expression of cell cycle progression as well as an enhanced mRNA expression of neuronal and glial differentiation markers. We further found a marked inactivation of the canonical Wnt pathway after the induction of cellular differentiation. Taken together, these data demonstrate the various molecular mechanisms taking place during the proliferation and early differentiation of enteric neural progenitor cells.

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#33: Single-cell RNA sequencing analysis of Zika virus infection in human stem cell-derived neuroprogenitor cells and cerebral organoids

Journal of the Pediatric Infectious Diseases Society ◽

10.1093/jpids/piab031.025 ◽

2021 ◽

Vol 10 (Supplement_2) ◽

pp. S11-S12

Author(s):

K E Ocwieja ◽

T K Hughes ◽

C C M Baker ◽

A C Stanton ◽

J M Antonucci ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Progenitor Cells ◽

Zika Virus ◽

Neural Progenitor Cells ◽

Neural Progenitor ◽

Human Stem Cell ◽

Zikv Infection ◽

Single Cell Sequencing ◽

Single Cell Rna Sequencing

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. It is also unclear why congenital ZIKV disease was not reported prior to the recent epidemics in French Polynesia and the Americas, despite evidence that Zika virus has actively circulated in parts of Africa and Asia since 1947 and 1966, respectively. Methods Due to advances in the stem cell-based technologies, we can now model ZIKV infections of the central nervous system in human stem cell-derived neural progenitor cells and cerebral organoids, which recapitulate complex 3-dimensional neural architecture. We apply Seq-Well — a simple, portable platform for massively parallel single-cell RNA sequencing — to characterize these neural models infected with ZIKV. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, thereby defining transcriptional features of both uninfected and infected cells. Results Although flavivirus RNAs lack a poly(A) tail, we present evidence that viral RNAs are specifically primed for reverse transcription at internal runs of adenosines, and that sequencing reads cover the entire non-polyadenylated viral genome. In neural progenitor cells, single cell sequencing reveals that while uninfected bystander cells strongly upregulate interferon pathway genes, these pathways are largely suppressed in cells infected with ZIKV within the same culture dish. Single cell sequencing identifies multiple cell types in our cerebral organoids including neural progenitor cells, intermediate progenitor cells, and neurons of varied maturity. Using this model, we find that neurons, not typically considered targets of ZIKV in the developing brain, contain high copy numbers of ZIKV genomes. It remains uncertain whether neurons are directly infected, or if infected neural progenitor cells differentiate into neurons, carrying virus with them. Notably, the neuronal bystander cell population shows limited interferon gene pathway upregulation compared to neural progenitors. Conclusions Overall, our work provides insight into the pathogenesis of ZIKV associated microcephaly, identifies potential new tropisms of ZIKV in the human brain, and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection.

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Clinical Outcomes of a Zika Virus Mother–Child Pair Cohort in Spain

Pathogens ◽

10.3390/pathogens9050352 ◽

2020 ◽

Vol 9 (5) ◽

pp. 352 ◽

Cited By ~ 1

Author(s):

Antoni Soriano-Arandes ◽

Marie Antoinette Frick ◽

Milagros García López-Hortelano ◽

Elena Sulleiro ◽

Carlota Rodó ◽

...

Keyword(s):

Zika Virus ◽

Adverse Outcomes ◽

Zikv Infection ◽

Child Pair ◽

Referral Hospitals ◽

Congenital Zika Syndrome ◽

Endemic Areas ◽

Neurodevelopmental Abnormalities ◽

Congenital Microcephaly

Background: Zika virus (ZIKV) infection has been associated with congenital microcephaly and other neurodevelopmental abnormalities. There is little published research on the effect of maternal ZIKV infection in a non-endemic European region. We aimed to describe the outcomes of pregnant travelers diagnosed as ZIKV-infected in Spain, and their exposed children. Methods: This prospective observational cohort study of nine referral hospitals enrolled pregnant women (PW) who travelled to endemic areas during their pregnancy or the two previous months, or those whose sexual partners visited endemic areas in the previous 6 months. Infants of ZIKV-infected mothers were followed for about two years. Results: ZIKV infection was diagnosed in 163 PW; 112 (70%) were asymptomatic and 24 (14.7%) were confirmed cases. Among 143 infants, 14 (9.8%) had adverse outcomes during follow-up; three had a congenital Zika syndrome (CZS), and 11 other potential Zika-related outcomes. The overall incidence of CZS was 2.1% (95%CI: 0.4–6.0%), but among infants born to ZIKV-confirmed mothers, this increased to 15.8% (95%CI: 3.4–39.6%). Conclusions: A nearly 10% overall risk of neurologic and hearing adverse outcomes was found in ZIKV-exposed children born to a ZIKV-infected traveler PW. Longer-term follow-up of these children is needed to assess whether there are any later-onset manifestations.

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Anti-Zika virus RNAi in neural progenitor cells

Cell Research ◽

10.1038/s41422-019-0158-3 ◽

2019 ◽

Vol 29 (4) ◽

pp. 261-262 ◽

Cited By ~ 2

Author(s):

Xuping Xie ◽

Pei-Yong Shi

Keyword(s):

Progenitor Cells ◽

Zika Virus ◽

Neural Progenitor Cells ◽

Neural Progenitor

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TRiC/CCT Complex, a Binding Partner of NS1 Protein, Supports the Replication of Zika Virus in Both Mammalians and Mosquitoes

Viruses ◽

10.3390/v12050519 ◽

2020 ◽

Vol 12 (5) ◽

pp. 519

Author(s):

Yuchen Wang ◽

Ryuta Uraki ◽

Jesse Hwang ◽

Erol Fikrig

Keyword(s):

Mass Spectrometry ◽

Zika Virus ◽

Mammalian Cells ◽

Host Factors ◽

Ns1 Protein ◽

Zikv Infection ◽

Binding Partner ◽

Promising Therapeutic Target ◽

Critical Components ◽

Congenital Microcephaly

Mosquito-borne Zika virus (ZIKV) can cause congenital microcephaly and Guillain–Barré syndrome, among other symptoms. Specific treatments and vaccines for ZIKV are not currently available. To further understand the host factors that support ZIKV replication, we used mass spectrometry to characterize mammalian proteins that associate with the ZIKV NS1 protein and identified the TRiC/CCT complex as an interacting partner. Furthermore, the suppression of CCT2, one of the critical components of the TRiC/CCT complex, inhibited ZIKV replication in both mammalian cells and mosquitoes. These results highlight an important role for the TRiC/CCT complex in ZIKV infection, suggesting that the TRiC/CCT complex may be a promising therapeutic target.

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Cell cycle progression is required for nuclear migration of neural progenitor cells

Brain Research ◽

10.1016/j.brainres.2006.03.042 ◽

2006 ◽

Vol 1088 (1) ◽

pp. 57-67 ◽

Cited By ~ 36

Author(s):

Masaki Ueno ◽

Kei-ichi Katayama ◽

Hirofumi Yamauchi ◽

Hiroyuki Nakayama ◽

Kunio Doi

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Cell Cycle Progression ◽

Neural Progenitor Cells ◽

Nuclear Migration ◽

Neural Progenitor ◽

Cycle Progression

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Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

10.1101/476440 ◽

2018 ◽

Cited By ~ 1

Author(s):

Michael F. Wells ◽

Max R. Salick ◽

Federica Piccioni ◽

Ellen J. Hill ◽

Jana M. Mitchell ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Zika Virus ◽

Large Scale ◽

Neural Progenitor Cells ◽

Neural Progenitor ◽

Autism Spectrum ◽

Host Factors ◽

Protein Markers ◽

Genome Wide

SUMMARYNeural progenitor cells (NPCs) are essential to brain development and their dysfunction is linked to several disorders, including autism, Zika Virus Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements with stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe humanStem cell-derivedNGN2-acceleratedProgenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, are proliferative, multipotent, and like other human NPCs, are susceptible to Zika-mediated death. We further demonstrate SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our studies expand knowledge of NPCs by identifying known and novel Zika host factors, as well as new regulators of NPC proliferation validated by re-identification of the autism spectrum genePTEN.

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Zika virus induces mitotic catastrophe in human neural progenitors by triggering unscheduled mitotic entry in the presence of DNA damage while functionally depleting nuclear PNKP

10.1101/2021.08.27.458001 ◽

2021 ◽

Author(s):

Malgorzata Rychlowska ◽

Abigail Agyapong ◽

Michael Weinfeld ◽

Luis M Schang

Keyword(s):

Dna Damage ◽

Zika Virus ◽

Cortical Neurons ◽

Genome Stability ◽

Neural Progenitor Cells ◽

Cellular Level ◽

Mitotic Catastrophe ◽

Zikv Infection ◽

Mitotic Entry ◽

Checkpoint Kinases

Vertical transmission of Zika virus (ZIKV) leads with high frequency to congenital ZIKV syndrome (CZS), whose worse outcome is microcephaly. However, the mechanisms of congenital ZIKV neurodevelopmental pathologies, including direct cytotoxicity to neural progenitor cells (NPC), placental insufficiency, and immune responses, remain incompletely understood. At the cellular level, microcephaly typically results from death or insufficient proliferation of NPC or cortical neurons. NPCs replicate fast, requiring efficient DNA damage responses to ensure genome stability. Like congenital ZIKV infection, mutations in the polynucleotide 5’-kinase 3’-phosphatase (PNKP) gene, which encodes a critical DNA damage repair enzyme, results in recessive syndromes often characterized by congenital microcephaly with seizures (MCSZ). We thus tested whether there were any links between ZIKV and PNKP. Here we show that a PNKP phosphatase inhibitor inhibits ZIKV replication. PNKP relocalized from the nucleus to the cytoplasm in infected cells, co-localizing with the marker of ZIKV replication factories (RF) NS1 and resulting in functional nuclear PNKP depletion. Although infected NPC accumulated DNA damage, they failed to activate the DNA damage checkpoint kinases Chk1 and Chk2. ZIKV also induced activation of cytoplasmic CycA/CDK1 complexes, which trigger unscheduled mitotic entry. Inhibition of CDK1 activity inhibited ZIKV replication and the formation of RF, supporting a role of cytoplasmic CycA/CDK1 in RF morphogenesis. In brief, ZIKV infection induces mitotic catastrophe resulting from unscheduled mitotic entry in the presence of DNA damage. PNKP and CycA/CDK1 are thus host factors participating in ZIKV replication in NPC, and probably pathogenesis.

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