22: Identifying zika virus (ZIKV)-silenced gene targets in the placenta at a transcriptome-wide scale using AGO-clip sequencing technology

Abstract

OBJECTIVE: We and others have recently demonstrated that the placental trophoblast serves as a reservoir for viral proliferation throughout pregnancy. How ZIKV evades normal endogenous mechanisms of viral suppression in the placenta is poorly understood. Based on established evidence that placenta-specific miRNAs (such as C19MC) serve as potent gene silencers by binding the 3' untranslated region (3'UTR), we hypothesized that identification of disrupted silenced placental miRNA targets following ZIKV infection at a transcriptome wide scale would elucidate means of evasion and potential therapeutic targets. STUDY DESIGN: Argonaute protein cross-linking immunoprecipitation coupled to high-throughput sequencing (AGO-CLIP-seq) served to directly identify targets of miRNAs by mapping AGO-RNA interactions. Using the Illumina TruSeq small RNA platform, we performed AGO-CLIP-Seq from primary human placental tissue (n=9). Analytics were completed on customized mapping pipelines, and annotated against human assemblies (Gencode). In validation experiments, we compared loss of silenced gene expression in isolated trophoblasts from n=10 unrelated donors infected in vitro with 1x105 pfu ZIKV or mock controls. RESULTS: The approach and results of the AGO-CLIP-seq analyses are shown in Fig. 1A. Consistent with placental-specific patterning, AGO-miRNA loading demonstrates a clear predominance (w28% of total expression) of C19MC placental-specific miRNA coverage, which are significantly enriched and account for 25% (115/447) of unique 3' UTR targets (Fig. 1B). In 10 unrelated subjects, we observed evidence of significantly disrupted miRNA-mediated gene silencing following infection of placental trophoblasts with ZIKV (Fig. 1C). Given their role in regulating apoptosis (CASP3) and fetal growth (IGFBP3), we find several to be of likely significance. CONCLUSION: This study is a novel approach to identify targets of viral mediated suppression, including several unique miRNA targets that may play a role in allowing ZIKV to infect placental trophoblastse. We speculate that miRNA mediated RNA control is a fundamental, rapid, molecular means of regulating the transcriptome in congenital viral infections, and our findings offer potential novel therapeutic targets.