microRNAs Control Antiviral Immune Response, Cell Death and Chemotaxis Pathways in Human Neuronal Precursor Cells (NPCs) during Zika Virus Infection

Abstract

Viral infections have always been a serious burden to public health, increasing morbidity and mortality rates worldwide. Zika virus (ZIKV) is a flavivirus transmitted by the Aedes aegypti vector and the causative agent of severe fetal neuropathogenesis and microcephaly. The virus crosses the placenta and reaches the fetal brain, mainly causing the death of neuronal precursor cells (NPCs), glial inflammation, and subsequent tissue damage. Genetic differences, mainly related to the antiviral immune response and cell death pathways greatly influence the susceptibility to infection. These components are modulated by many factors, including microRNAs (miRNAs). MiRNAs are small noncoding RNAs that regulate post-transcriptionally the overall gene expression, including genes for the neurodevelopment and the formation of neural circuits. In this context, we investigated the pathways and target genes of miRNAs modulated in NPCs infected with ZIKV. We observed downregulation of miR-302b, miR-302c and miR-194, whereas miR-30c was upregulated in ZIKV infected human NPCs in vitro. The analysis of a public dataset of ZIKV-infected human NPCs evidenced 262 upregulated and 3 downregulated genes, of which 142 were the target of the aforementioned miRNAs. Further, we confirmed a correlation between miRNA and target genes affecting pathways related to antiviral immune response, cell death and immune cells chemotaxis, all of which could contribute to the establishment of microcephaly and brain lesions. Here, we suggest that miRNAs target gene expression in infected NPCs, directly contributing to the pathogenesis of fetal microcephaly.

Keywords: ZIKV; cell death; chemotaxis; inflammation; microRNA.

Figure 1

ZIKV infects human NPCs. Human NPCs were (A) infected or not with ZIKV (MOI = 1). 10× magnification. After 48 h post infection, they were evaluated for (B) viral copy numbers by qPCR (n = 14) and (C) PFU. (n = 8). In (D) ZIKV infection was confirmed by immunofluorescence with anti-envelope flavivirus antibody at 20× magnification (n = 3). Graphs represent three independent experiments. Unpaired t-test.

Figure 2

ZIKV modulates miRNA profile in human NPCs. (A) Human NPCs were infected or not with ZIKV (MOI = 1). After 48 h we evaluated the miRNA profile by qPCR array. Volcano Plot, white dots—unaltered; blue dots—downregulated; red dots—upregulated. Three independent experiments. Each plate represents a pool of samples (n = 3) from each independent experiment. (B) Table of downregulated miRNAs; (C) Table of upregulated miRNAs. (D) Interactome of modulated miRNAs and their predicted targets in hiNPCs from public RNA-sequencing.

Figure 3

ZIKV downregulates miRNAs increasging cell death. (A) Heat-map, (B) bubble plot of gene ontology, and (C) volcano plot of public RNA-sequencing from hiNPCs (n = 3). 31,040 variables. (D) Quantification of IL−1β by CBA in supernatant of CTRL or ZIKV infected human NPCs after 48 h post infection (n = 12). Graphs represent three independent experiments. Unpaired t-test.

Figure 4

ZIKV downregulates miRNAs to recruit immune cells. (A) Heat-map, (B) Bubble plot of gene ontology, and (C) volcano plot of public RNA-sequencing from hiNPCs (n = 3). 31,040 variables. (D) Quantification of IL−8 by CBA in supernatant of CTRL or ZIKV infected human NPCs after 48 h post infection (n = 12). Graphs represent three independent experiments. Unpaired t-test.

Figure 5

ZIKV downregulates miRNAs to modulate antiviral immune response. (A) Heat-map, (B) Bubble plot of gene ontology, and (C) volcano plot of public RNA-sequencing from hiNPCs (n = 3). 31,040 variables. (D) Gene expression of MXD1, and ISG20 by qPCR (n = 8), and (E) Quantification of TNF−α, and IL−6 by CBA in supernatant of CTRL or ZIKV infected human NPCs after 48 h post infection (n = 12). Graphs represent three independent experiments. Unpaired t-test.

Figure 6

ZIKV downregulates miRNAs to increase ubiquitination and antigen presentation. (A) Heat-map, (B) Bubble plot of gene ontology, and (C) volcano plot of public RNA-sequencing from hiNPCs (n = 3). 31,040 variables.