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Zika virus E protein alters the properties of human fetal neural stem cells by modulating microRNA circuitry

Cell Death & Differentiationvolume 25pages18371854 (2018) | Download Citation

Abstract

Zika virus (ZV) infects neural stem cells (NSCs) and causes quiescence in NSCs, reducing the pool of brain cells, leading to microcephaly. Despite conscientious efforts, the molecular mechanisms for ZV-mediated effects on NSCs lack clarity. This study aimed to explore the underlying mechanisms for ZV-mediated induction of quiescence in the primary cultures of human fetal neural stem cells (fNSCs). We demonstrate that expression of ZV envelope (E) protein displays maximum quiescence in human fNSCs by accumulating cells in the G0/G1 phase of the cell cycle as compared to other non-structural proteins, viz. NS2A, NS4A and NS4B. E protein induces immature differentiation by induction of pro-neuronal genes in proliferating fNSCs, induces apoptosis in differentiating fNSCs 3 days post differentiation, and disrupts migration of cells from differentiating neurospheres. In utero electroporation of mouse brain with E protein shows drastic downregulation of proliferating cells in ventricular and subventricular zone regions. Global microRNA sequencing suggests that E protein modulates miRNA circuitry. Among differentially expressed miRNAs, we found 14 upregulated and 11 downregulated miRNAs. Mir-204-3p and mir-1273g-3p directly regulate NOTCH2 and PAX3 expression, respectively, by binding to their 3′UTR. Bioinformatic analysis using GO analysis for the targets of differentially expressed miRNAs revealed enrichment of cell cycle and developmental processes. Furthermore, WNT, CCKR, PDGF, EGF, p53, and NOTCH signaling pathways were among the top enriched pathways. Thus, our study provides evidence for the involvement of ZV E protein and novel insights into the molecular mechanism through identification of miRNA circuitry.

Art work depicting the effect of Zika virus E protein on human fetal neural stem cells.

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Acknowledgements

Technical assistance from Mr. DL Meena and Mr. Naushad Alam of NBRC, India, is acknowledged. Ms. Bindu Singh and Ms. Priyanka S Kshtriya helped in initial assays. Junior and Senior Research Fellowship to Ms. Reshma Bhagat from CSIR, New Delhi, India, is greatly acknowledged. Expression vectors for the four ZV proteins were kind gift from Dr. Shyamala Mani, IISc, India (These were possible due to financial support from CEFIPRA (4903-02) awarded to P. Gressens (INSERM) and S. Mani). Financial support for the study from NBRC core funds to PS is greatly acknowledged. The authors also wish to acknowledge the support of the facilities provided under the Biotechnology Information System Network (BTISNET) grant, DBT, India, and Distributed Information Centre at NBRC, Manesar, India.

Author contributions

RB designed and performed the experiments, analyzed the data, and wrote the manuscript. BP did the bioinformatics analysis of small RNA sequencing data. SN did qPCR experiments for revised version. NA performed and analyzed the in utero electroporation experiments. JS designed, analyzed and interpreted the in utero electroporation experiments. YKA provided inputs and helped in writing the manuscript. SM designed the vectors, provided the initial inputs, PS designed the study, helped in analyzing and interpretation of data and approved the manuscript.

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Affiliations

  1. Department of Cellular and Molecular Neuroscience, Neurovirology Section, National Brain Research Centre, Manesar, Gurgaon, Haryana, India

    • Reshma Bhagat
    • , Bharat Prajapati
    • , Sonia Narwal
    • , Yogita K. Adlakha
    •  & Pankaj Seth
  2. Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh, India

    • Nitin Agnihotri
    •  & Jonaki Sen
  3. Curadev Pharma Pvt. Ltd, B87 Sector 83, Noida, Uttar Pradesh, India

    • Shyamala Mani
  4. INSERM, U1141, Hôpital Robert Debré, Paris, France

    • Shyamala Mani

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The authors declare that they have no conflict of interest.

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Correspondence to Pankaj Seth.

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https://doi.org/10.1038/s41418-018-0163-y