Contribution of Dicer, RNAi and regulatory RNAsRNAi and microRNAs to the antiviral response against Zika virus in trophoblast cells

The Zika virus (ZIKV), a mosquito-borne arbovirus, represents a significant public health concern, particularly due to its capacity for maternal-fetal transmission during pregnancy and its association with severe congenital malformations in newborns. Trophoblast cells from the placenta exhibit unique immunological features, including the constitutive production of type III interferons and the exclusive expression of a microRNA (miRNA) cluster known as C19MC, located on chromosome 19. This project aims to elucidate the role of the RNA interference (RNAi) and the miRNA pathways in the antiviral response of human trophoblast cells to ZIKV infection. Particular emphasis will be placed on the role of Dicer and Drosha, as well as the placenta-specific C19MC cluster. Using JAR choriocarcinoma cells and trophoblast stem (TS) cells, Dicer and Drosha expression will be silenced by siRNA or knocked-out via CRISPR-Cas9. The replication of ZIKV will be investigated in these cell lines to determine the functional contribution of these RNAi components. The role of the C19MC miRNA cluster will be explored using a JAR cell line where C19MC has been deleted by CRISPR-Cas9. The impact of C19MC loss on ZIKV replication will be further analyzed, providing insights into the role of placenta-specific miRNAs in antiviral defense. To determine the contribution of RNAi pathway, small RNA sequencing will be performed on total and AGO2-immunoprecipitated RNA from infected cells to detect virus-derived siRNAs (vsiRNAs). The functional activity of vsiRNAs will be validated by a dual fluorescent reporter system containing viral sequences. Concurrently, miRNA profiles will be analyzed to determine whether ZIKV infection alters specific miRNA subsets. In addition, RNA sequencing (RNA-seq) will identify genes deregulated by ZIKV infection. Cross-analysis of miRNA and mRNA data will help uncover potential miRNA target genes. Gene networks and cellular pathways deregulated during ZIKV infection will be analyzed and candidate genes will be validated, and their importance validated through siRNA-mediated knock-down. Another goal of the project will be to identify host proteins bound to ZIKV RNA, by using two complementary proteomic approaches: the immunoprecipitation using the dsRNA-specific J2 antibody, and a direct RNA pulldown using antisense biotinylated probes. The presence of Dicer, Drosha, and known dsRNA-binding proteins (e.g., TRBP, PACT, PKR) will be detected by western blot analysis. To get a comprehensive picture of ZIKV RNA-associated proteins, the samples will also be analyzed by mass spectrometry. Candidate proteins will be validated by western blot, confirming known factors and revealing new interactions that may contribute to the antiviral response. This project will determine the respective roles of the RNAi machinery and placental miRNAs in trophoblast defense against ZIKV and identify new host factors interacting with viral RNA, opening avenues for future antiviral strategies.