Amy H. Buck

The evolution of RNAi as a defence against viruses and transposable elements.

RNA interference (RNAi) is an important defence against viruses and transposable elements (TEs). RNAi not only protects against viruses by degrading viral RNA, but hosts and viruses can also use RNAi to manipulate each others gene expression, and hosts can encode microRNAs that target viral sequences. In response, viruses have evolved a myriad of adaptations to suppress and evade RNAi. RNAi can also protect cells against TEs, both by degrading TE transcripts and by preventing TE expression through heterochromatin formation. The aim of our review is to summarize and evaluate the current data on the evolution of these RNAi defence mechanisms. To this end, we also extend a previous analysis of the evolution of genes of the RNAi pathways. Strikingly, we find that antiviral RNAi genes, anti-TE RNAi genes and viral suppressors of RNAi all evolve rapidly, suggestive of an evolutionary arms race between hosts and parasites. Over longer time scales, key RNAi genes are repeatedly duplicated or lost across the metazoan phylogeny, with important implications for RNAi as an immune defence.

Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity

In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.

Post-transcriptional regulation of miR-27 in murine cytomegalovirus infection

In mammals, microRNAs (miRNAs) can play diverse roles in viral infection through their capacity to regulate both host and viral genes. Recent reports have demonstrated that specific miRNAs change in expression level upon infection and can impact viral production and infectivity. It is clear that miRNAs are an integral component of viral-host interactions, and it is likely that both host and virus contain mechanisms to regulate miRNA expression and/or activity. To date, little is known about the mechanisms by which miRNAs are regulated in viral infection. Here we report the rapid down-regulation of miR-27a in multiple mouse cell lines as well as primary macrophages upon infection with the murine cytomegalovirus. Down-regulation of miR-27a occurs independently from two other miRNAs, miR-23a and miR-24, located within the same genomic cluster, and analysis of pri-miRNA levels suggest that regulation occurs post-transcriptionally. miR-27b, a close homolog of miR-27a (20/21 nucleotide identity), also decreases upon infection, and we demonstrate that both miR-27a and miR-27b exert an antiviral function upon over-expression. Drug sensitivity experiments suggest that virus entry is not sufficient to induce the down-regulation of miR-27 and that the mechanism requires synthesis of RNA. Altogether, our findings indicate that miR-27a and miR-27b have antiviral activity against MCMV, and that either the virus or the host encodes molecule(s) for regulating miR-27 accumulation, most likely by inducing the rapid decay of the mature species.

The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper

ABSTRACT The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge – of the nature of EV(-RNA)s and of how to effectively and reliably study them – currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

Induction of IL-4Rα–dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4–driven murine macrophage proliferation in vivo

Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4-receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4-driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα-induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4-driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.

Combined agonist-antagonist genome-wide functional screening identifies broadly active antiviral microRNAs

Although the functional parameters of microRNAs (miRNAs) have been explored in some depth, the roles of these molecules in viral infections remain elusive. Here we report a general method for global analysis of miRNA function that compares the significance of both overexpressing and inhibiting each mouse miRNA on the growth properties of different viruses. Our comparative analysis of representatives of all three herpesvirus subfamilies identified host miRNAs with broad anti- and proviral properties which extend to a single-stranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual host-encoded miRNA regulates multiple pathways required and/or activated by viruses, including PI3K/AKT and ERK/MAPK signaling, oxidative stress signaling, and prostaglandin synthesis. Global miRNA expression analysis further demonstrated that the miR-199a/miR-214 cluster is down-regulated in both murine and human cytomegalovirus infection and manifests similar antiviral properties in mouse and human cells. Overall, we report a general strategy for examining the contributions of individual host miRNAs in viral infection and provide evidence that these molecules confer broad inhibitory potential against multiple viruses.

Murine cytomegalovirus encodes a miR-27 inhibitor disguised as a target

Individual microRNAs (miRNAs) are rapidly down-regulated during conditions of cellular activation and infection, but factors mediating miRNA turnover are poorly understood. Infection of mouse cells with murine cytomegalovirus (MCMV) induces the rapid down-regulation of an antiviral cellular miRNA, miR-27. Here, we identify a transcript produced by MCMV that binds to miR-27 and mediates its degradation. UV-crosslinking and high-throughput sequencing [CRAC (UV-crosslinking and analysis of cDNA)] identified MCMV RNA segments associated with the miRNA-binding protein Argonaute 2 (Ago2). A cluster of hits mapped to a predicted miR-27-binding site in the 3′UTR of the previously uncharacterized ORF, m169. The expression kinetics of the m169 transcript correlated with degradation of miR-27 during infection, and m169 expression inhibited miR-27 functional activity in a reporter assay. siRNA knockdown of m169 demonstrated its requirement for miR-27 degradation following infection and did not affect other host miRNAs. Substitution of the miR-27-binding site in m169 to create complementarity to a different cellular miRNA, miR-24, resulted in down-regulation of only miR-24 following infection. The m169 transcript is cytoplasmic, capped, polyadenylated, and interacts with miRNA-27 through seed pairing: characteristic features of the normal messenger RNA (mRNA) targets of miRNAs. This virus–host interaction reveals a mode of miRNA regulation in which a mRNA directs the degradation of a miRNA. We speculate that RNA-mediated miRNA degradation could be a more general viral strategy for manipulating host cells.

Discrete Clusters of Virus-Encoded MicroRNAs Are Associated with Complementary Strands of the Genome and the 7.2-Kilobase Stable Intron in Murine Cytomegalovirus

ABSTRACT The prevalence and importance of microRNAs (miRNAs) in viral infection are increasingly relevant. Eleven miRNAs were previously identified in human cytomegalovirus (HCMV); however, miRNA content in murine CMV (MCMV), which serves as an important in vivo model for CMV infection, has not previously been examined. We have cloned and characterized 17 novel miRNAs that originate from at least 12 precursor miRNAs in MCMV and are not homologous to HCMV miRNAs. In parallel, we applied a computational analysis, using a support vector machine approach, to identify potential precursor miRNAs in MCMV. Four of the top 10 predicted precursor sequences were cloned in this study, and the combination of computational and cloning analysis demonstrates that MCMV has the capacity to encode miRNAs clustered throughout the genome. On the basis of drug sensitivity experiments for resolving the kinetic class of expression, we show that the MCMV miRNAs are both early and late gene products. Notably, the MCMV miRNAs occur on complementary strands of the genome in specific regions, a feature which has not previously been observed for viral miRNAs. One cluster of miRNAs occurs in close proximity to the 5′ splice site of the previously identified 7.2-kb stable intron, implying a variety of potential regulatory mechanisms for MCMV miRNAs.

Parasite-Derived MicroRNAs in Host Serum As Novel Biomarkers of Helminth Infection

Background MicroRNAs (miRNAs) are a class of short non-coding RNA that play important roles in disease processes in animals and are present in a highly stable cell-free form in body fluids. Here, we examine the capacity of host and parasite miRNAs to serve as tissue or serum biomarkers of Schistosoma mansoni infection. Methods/Principal Findings We used Exiqon miRNA microarrays to profile miRNA expression in the livers of mice infected with S. mansoni at 7 weeks post-infection. Thirty-three mouse miRNAs were differentially expressed in infected compared to naïve mice (>2 fold change, p<0.05) including miR-199a-3p, miR-199a-5p, miR-214 and miR-21, which have previously been associated with liver fibrosis in other settings. Five of the mouse miRNAs were also significantly elevated in serum by twelve weeks post-infection. Sequencing of small RNAs from serum confirmed the presence of these miRNAs and further revealed eleven parasite-derived miRNAs that were detectable by eight weeks post infection. Analysis of host and parasite miRNA abundance by qRT-PCR was extended to serum of patients from low and high infection sites in Zimbabwe and Uganda. The host-derived miRNAs failed to distinguish uninfected from infected individuals. However, analysis of three of the parasite-derived miRNAs (miR-277, miR-3479-3p and bantam) could detect infected individuals from low and high infection intensity sites with specificity/sensitivity values of 89%/80% and 80%/90%, respectively. Conclusions This work identifies parasite-derived miRNAs as novel markers of S. mansoni infection in both mice and humans, with the potential to be used with existing techniques to improve S. mansoni diagnosis. In contrast, although host miRNAs are differentially expressed in the liver during infection their abundance levels in serum are variable in human patients and may be useful in cases of extreme pathology but likely hold limited value for detecting prevalence of infection.