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Cultured primary human trophoblasts (PHT), derived from term placentas, are relatively resistant to infection by diverse viruses. The resistance can be conferred to non-trophoblastic cells by pre-exposing them to medium that was conditioned by PHT cells. This antiviral effect is mediated, at least in part, by microRNAs (miRNA) expressed from the chromosome 19 microRNA cluster (C19MC). Recently we showed that PHT cells and cells pre-exposed to PHT medium are also resistant to infection by Zika virus (ZIKV), an effect mediated by the constitutive release of the type III interferons (IFN) IFN lambda-1 and IFN lambda-2 in trophoblastic medium. We hypothesized that trophoblastic C19MC miRNA are active against ZIKV, and assessed the interaction of this pathway with IFN lambda-1 - mediated resistance.Term PHT cells were cultured using standard techniques. An osteosarcoma cell line (U2OS) was used as non-trophoblastic cells, which were infected with either ZIKV or vesicular stomatitis virus (VSV). Trophoblastic extracellular vesicles (EVs) were produced by gradient ultracentrifugation. RT-qPCR was used to determine viral infection, cellular or medium miRNA levels and the expression of interferon-stimulated genes.We showed that C19MC miRNA attenuate infection of U2OS cells by ZIKV, and that C19MC miRNA or exosomes that contain C19MC miRNA did not influence the type III IFN pathway. Similarly, cell exposure to recombinant IFN lambda-1 had no effect on miRNA expression, and these pathways did not exhibit synergistic interaction.PHT cells exert antiviral activity by at least two independent mechanisms, mediated by C19MC miRNA and by type III IFNs.

Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs.We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs.We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts.When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication.

While cytomegalovirus (CMV) infections are often limited in host range by lengthy coevolution with a single host species, a few CMVs are known to deviate from this rule. For example, rhesus macaque CMV (RhCMV), a model for human CMV (HCMV) pathogenesis and vaccine development, can replicate in human cells, as well as in rhesus cells. Both HCMV and RhCMV encode species-specific antagonists of the broadly acting host cell restriction factor protein kinase R (PKR). Although the RhCMV antagonist of PKR, rTRS1, has very limited activity against human PKR, here, we show it is essential for RhCMV replication in human cells because it prevents human PKR from phosphorylating the translation initiation factor eIF2α, thereby allowing continued translation and viral replication. Although rTRS1 is necessary for RhCMV replication, it is not sufficient to rescue replication of HCMV lacking its own PKR antagonists in human fibroblasts. However, overexpression of rTRS1 in human fibroblasts enabled HCMV expressing rTRS1 to replicate, indicating that elevated levels or early expression of a weak antagonist can counteract a resistant restriction factor like human PKR. Exploring potential mechanisms that might allow RhCMV to replicate in human cells revealed that RhCMV makes no less double-stranded RNA than HCMV. Rather, in human cells, RhCMV expresses rTRS1 at levels 2 to 3 times higher than those of the HCMV-encoded PKR antagonists during HCMV infection. These data suggest that even a modest increase in expression of this weak PKR antagonist is sufficient to enable RhCMV replication in human cells. IMPORTANCE Rhesus macaque cytomegalovirus (RhCMV) offers a valuable model for studying congenital human cytomegalovirus (HCMV) pathogenesis and vaccine development. Therefore, it is critical to understand variations in how each virus infects and affects its host species to be able to apply insights gained from the RhCMV model to HCMV. While HCMV is capable only of infecting cells from humans and very closely related species, RhCMV displays a wider host range, including human as well as rhesus cells. RhCMV expresses an antagonist of a broadly acting antiviral factor present in all mammalian cells, and its ability to counter both the rhesus and human versions of this host factor is a key component of RhCMV's ability to cross species barriers. Here, we examine the molecular mechanisms that allow this RhCMV antagonist to function against a human restriction factor.

During the past decade, various types of small non-coding RNAs were found to be expressed in all kingdoms and phyla of life. Intense research efforts have begun to shed light on their biological functions, although much remains to be determined in order to fully characterize their scope of biological action. Typically, small RNAs provide sequence specificity to a protein complex that is driven to silence a long target RNA. MicroRNAs (miRNAs) are small RNAs that are coded in the genome of most eukaryotes, and contribute to the cellular identity by regulating cell-specific gene networks by translational repression or degradation of mRNA. These effects commonly fine-tune gene expression associated with developmental or environmental cues. Different cell types can be characterized by their distinctive cellular miRNA landscape. The human placenta expresses a unique set of miRNAs, a high proportion of which is derived from a large cluster located on chromosome 19, (termed chromosome 19 miRNA cluster, or C19MC). Interestingly, a fraction of these placenta-enriched miRNAs are released to the extracellular environment through exosomes that were recently found to induce an antiviral immunity. In this review, we explore relevant placental viral infections and discuss the antiviral role of exosome-packaged placental C19MC miRNAs in this context.

Placental trophoblasts form the interface between the fetal and maternal environments and serve to limit the maternal–fetal spread of viruses. Here we show that cultured primary human placental trophoblasts are highly resistant to infection by a number of viruses and, importantly, confer this resistance to nonplacental recipient cells by exosome-mediated delivery of specific microRNAs (miRNAs). We show that miRNA members of the chromosome 19 miRNA cluster, which are almost exclusively expressed in the human placenta, are packaged within trophoblast-derived exosomes and attenuate viral replication in recipient cells by the induction of autophagy. Together, our findings identify an unprecedented paracrine and/or systemic function of placental trophoblasts that uses exosome-mediated transfer of a unique set of placental-specific effector miRNAs to directly communicate with placental or maternal target cells and regulate their immunity to viral infections.

Mechanisms to protect against viral infections are crucial during pregnancy as maternal-fetal transmission can have serious pathological outcomes, including fetal infection and its sequelae, such as growth restriction, birth defects, and/or fetal death. The trophoblast forms the interface between the feto-placental unit and the maternal blood, and is therefore a critical physical and immunological barrier to restrict the spread of pathogens into the fetal microenvironment. Recently, we found that primary human placental trophoblast (PHT) cells are highly resistant to infection by diverse viruses. In this study, we also found that conditioned medium from PHT cell cultures transferred viral resistance to nonplacental recipient cells, suggesting that a component secreted by trophoblasts and present within the conditioned medium is responsible for this antiviral effect. We found that specific miRNAs from a unique primate- and placental-specific locus?the C19MC (chromosome 19 miRNA cluster)?are packaged within exosomes produced by PHT cells and confer viral resistance in nonplacental recipient cells. In addition to conveying viral resistance, we found that PHT-derived exosomes and select miRNA members of the C19MC family strongly induce autophagy, which is involved in recipient cell viral resistance. Our findings establish an exciting and novel mechanism by which placental trophoblasts exploit exosome-dependent transfer of placental-specific miRNAs to influence autophagic induction and antiviral immunity at the maternal?fetal interface.

Positioned at the maternal-fetal interface, the placenta generates regulatory signals and serves as a communication junction that governs pregnancy health. In addition to hormones, growth factors and diverse proteins, we and others have recently shown that small non-coding RNA molecules, including microRNAs (miRNAs), traffic among the maternalplacental-fetal compartments, and may have a central role in maternalfetal adaptation to homeostatic perturbations. Although common and unique types of miRNAs are expressed by the placenta during pregnancy, the function of placental miRNA species remains largely unknown. We recently showed that primary human trophoblasts are resistant to infection by a heterogeneous panel of viruses, and that this resistance can be partly conferred to non-trophoblast cells by exogenous expression of trophoblast-specific miRNAs, expressed from the chromosome 19 miRNA cluster (C19MC). This effect was not observed during cell infection by nonviral placental pathogens, including Listeria monocytogenes, and Toxoplasma gondii. Trophoblastic miRNAs can be released to the extracellular space within several types of vesicles, including cell fragments, apoptotic bodies, microvesicles, and nanovesicles (exosomes), or can be packaged with non-vesicular protein complexes, such as argonaute-2. To examine the trafficking of trophoblastic miRNAs to maternal-fetal tissues we engineered a transgenic mouse that expresses a 160 kb segment of human genomic DNA harboring the human C19MC cluster. We coupled this technology with RNAseq analysis of miRNA expression in triads of placenta, maternal plasma and fetal plasma from pregnant women at term. Together, these analyses shed new light on transplacental bidirectional miRNA trafficking patterns (Supported by NIH HD06589, HD075665, HD071707, AI081759, and the Pennsylvania Department of Health Research Formula Funds)