Profound downregulation of neural transcription factor Npas4 and Nr4a family in fetal mice neurons infected with Zika virus

Zika virus (ZIKV) infection of neurons leads to neurological complications and congenital malformations of the brain of neonates. To date, ZIKV mechanism of infection and pathogenesis is not entirely understood and different studies on gene regulation of ZIKV-infected cells have identified a dysregulation of inflammatory and stem cell maintenance pathways. MicroRNAs (miRNAs) are post-transcriptional regulators of cellular genes and they contribute to cell development in normal function and disease. Previous reports with integrative analyses of messenger RNAs (mRNAs) and miRNAs during ZIKV infection have not identified neurological pathway defects. We hypothesized that dysregulation of pathways involved in neurological functions will be identified by RNA profiling of ZIKV-infected fetal neurons. We therefore used microarrays to analyze gene expression levels following ZIKV infection of fetal murine neurons. We observed that the expression levels of transcription factors such as neural PAS domain protein 4 (Npas4) and of three members of the orphan nuclear receptor 4 (Nr4a) were severely decreased after viral infection. We confirmed that their downregulation was at both the mRNA level and at the protein level. The dysregulation of these transcription factors has been previously linked to aberrant neural functions and development. We next examined the miRNA expression profile in infected primary murine neurons by microarray and found that various miRNAs were dysregulated upon ZIKV infection. An integrative analysis of the differentially expressed miRNAs and mRNAs indicated that miR-7013-5p targets Nr4a3 gene. Using miRmimics, we corroborated that miR-7013-5p downregulates Nr4a3 mRNA and protein levels. Our data identify a profound dysregulation of neural transcription factors with an overexpression of miR-7013-5p that results in decreased Nr4a3 expression, likely a main contributor to ZIKV-induced neuronal dysfunction. Author summary Zika virus (ZIKV) is an emerging virus transmitted horizontally between humans through mosquito bites, and sexual intercourse generally inducing a mild disease. ZIKV is also transmitted vertically from mother-to-child producing congenital ZIKV syndrome (CZVS) in neonates. CZVS leads to severe microcephaly associated with neurological, ocular, musculoskeletal, genitourinary disorders and other disabilities. Although numerous studies have been performed on ZIKV infection of brain cells, we are still far from understanding how ZIKV infection leads to dysregulation of host genes, virus-induced cytopathicity and consequent pathology. Micro (mi)RNAs are small noncoding RNAs encoded and processed by the host cell. They regulate gene expression at the post-transcriptional level in a process called RNA interference (RNAi). Here, we evaluated the relationship between ZIKV infection and the level of mRNAs and miRNAs expressed in the cell. ZIKV infection of mouse embryo neurons downregulated several neural immediate-early genes (IEG). Moreover, we revealed that ZIKV infection led to aberrant regulation of several miRNAs, and identified one whose cognate target was a neural IEG. Our work identifies novel genes and miRNAs that are modulated upon ZIKV infection of fetal murine neurons, therefore linking neuronal dysfunction to transcription and the RNA interference pathway.

Automated summary

Zika virus (ZIKV) infection results in dysregulation of neural transcription factors and downregulation of microRNAs, specifically miR-7013-5p, leading to neuronal dysfunction. This study highlights the importance of the RNA interference pathway in ZIKV-induced pathology.