Zika virus (ZIKV) congenital infection evades double-stranded RNA detection and may persist in the placenta for the duration of pregnancy, without accompanying overt histopathologic inflammation. Understanding how viruses can persist and replicate in the placenta, without causing overt cellular or tissue damage, is fundamental to deciphering mechanisms of maternal-fetal vertical transmission.
Placenta-specific microRNAs (miRNAs) are believed to be a tenet of viral resistance at the maternal-fetal interface. We aimed to test the hypothesis that ZIKV functionally disrupts placental miRNAs, enabling viral persistence and fetal pathogenesis.
To test this hypothesis, we utilized orthogonal approaches in human and murine experimental models. In primary human trophoblast cultures (n=5 donor placentae), we performed high-throughput sequencing crosslinking and immunoprecipitation (AGO-HITS-CLIP) to identify any significant alterations in the functional loading of miRNAs and their targets onto the RNA-induced silencing complex (RISC). Trophoblasts from same-donors were split, and infected with a contemporary first-passage ZIKV strain HN16 (MOI=1 plaque forming unit per cell) or mock infected. To functionally cross-validate miRNA-mRNA interactions, we compared our AGO-HITS-CLIP results with an independent analysis of published (Lum et al., 2019) bulk RNA-seq data from human placental disc specimens (n=3 subjects; ZIKV-positive in 1, 2, or 3-trimester, CD45 cells sorted by flow-cytometry) compared to uninfected controls (n=2 subjects). To investigate the importance of these miRNA and RNAi networks in ZIKV pathogenesis, we utilized a gnotobiotic mouse model uniquely susceptible to ZIKV infection. We evaluate if small-molecule enhancement of miRNA and RNAi pathways with enoxacin influenced ZIKV pathogenesis (n=20 dams total yielding 187 fetal specimens). Lastly, placentae (n=14 total) from this mouse model were analyzed with Visium spatial transcriptomics (9,743 spatial transcriptomes) to identify potential ZIKV-associated alterations in immune microenvironments.
We found that ZIKV infection of primary human trophoblast cells led to an unexpected disruption of placental miRNA regulation networks. Compared to uninfected controls, ZIKV-infected placentae had significantly altered SLC12A8, SDK1, and VLDLR RISC-loading and transcript levels (-22; adjusted p-value<0.05, Wald-test with FDR correction q<0.05). In silico miRNA target analyses revealed 26 of 119 transcripts (22%) in the TGF-β Signaling Pathway were targeted by miRNAs which were found to be dysregulated following ZIKV infection in trophoblasts. In gnotobiotic mice, relative to mock controls, ZIKV-associated fetal pathogenesis included fetal growth restriction (p=0.036) and viral persistence in placental tissue (p=0.011). Moreover, spatial transcriptomics of murine placentae revealed that ZIKV-specific placental niches were defined by significant upregulation of complement cascade components and coordinated changes in TGF-β gene expression. Finally, treatment of ZIKV-infected mice with enoxacin abolished placental ZIKV persistence, rescued the associated fetal growth restriction, and ZIKV-associated transcriptional changes in placental immune microenvironments were no longer observed.
These results collectively suggest that (i) ZIKV infection and persistence is associated with functionally perturbed miRNA and RNAi pathways specifically related to immune regulation in placental microenvironments, and (ii) enhancement of placental miRNA and RNAi pathways in mice rescued ZIKV-associated pathogenesis, specifically persistence of viral transcripts in placental microenvironments and fetal growth restriction.

Copyright © 2023 Elsevier Inc. All rights reserved.