Broad-Spectrum Inhibition of Respiratory Virus Infection by MicroRNA Mimics Targeting p38 MAPK Signaling.

The majority of antiviral therapeutics target conserved viral proteins, however, this approach confers selective pressure on the virus and increases the probability of antiviral drug resistance. An alternative therapeutic strategy is to target the host-encoded factors that are required for virus infection, thus minimizing the opportunity for viral mutations that escape drug activity. MicroRNAs (miRNAs) are small noncoding RNAs that play diverse roles in normal and disease biology, and they generally operate through the post-transcriptional regulation of mRNA targets. We have previously identified cellular miRNAs that have antiviral activity against a broad range of herpesvirus infections, and here we extend the antiviral profile of a number of these miRNAs against influenza and respiratory syncytial virus. From these screening experiments, we identified broad-spectrum antiviral miRNAs that caused >75% viral suppression in all strains tested, and we examined their mechanism of action using reverse-phase protein array analysis. Targets of lead candidates, miR-124, miR-24, and miR-744, were identified within the p38 mitogen-activated protein kinase (MAPK) signaling pathway, and this work identified MAPK-activated protein kinase 2 as a broad-spectrum antiviral target required for both influenza and respiratory syncytial virus (RSV) infection.

Extracellular Vesicles from a Helminth Parasite Suppress Macrophage Activation and Constitute an Effective Vaccine for Protective Immunity.

Recent studies have demonstrated that many parasites release extracellular vesicles (EVs), yet little is known about the specific interactions of EVs with immune cells or their functions during infection. We show that EVs secreted by the gastrointestinal nematode Heligmosomoides polygyrus are internalized by macrophages and modulate their activation. EV internalization causes downregulation of type 1 and type 2 immune-response-associated molecules (IL-6 and TNF, and Ym1 and RELMα) and inhibits expression of the IL-33 receptor subunit ST2. Co-incubation with EV antibodies abrogated suppression of alternative activation and was associated with increased co-localization of the EVs with lysosomes. Furthermore, mice vaccinated with EV-alum generated protective immunity against larval challenge, highlighting an important role in vivo. In contrast, ST2-deficient mice are highly susceptible to infection, and they are unable to clear parasites following EV vaccination. Hence, macrophage activation and the IL-33 pathway are targeted by H. polygyrus EVs, while neutralization of EV function facilitates parasite expulsion.

Plasmalogen enrichment in exosomes secreted by a nematode parasite versus those derived from its mouse host: implications for exosome stability and biology.

Extracellular vesicles (EVs) mediate communication between cells and organisms across all 3 kingdoms of life. Several reports have demonstrated that EVs can transfer molecules between phylogenetically diverse species and can be used by parasites to alter the properties of the host environment. Whilst the concept of vesicle secretion and uptake is broad reaching, the molecular composition of these complexes is expected to be diverse based on the physiology and environmental niche of different organisms. Exosomes are one class of EVs originally defined based on their endocytic origin, as these derive from multivesicular bodies that then fuse with the plasma membrane releasing them into the extracellular environment. The term exosome has also been used to describe any small EVs recovered by high-speed ultracentrifugation, irrespective of origin since this is not always well characterized. Here, we use comparative global lipidomic analysis to examine the composition of EVs, which we term exosomes, that are secreted by the gastrointestinal nematode, Heligmosomoides polygyrus, in relation to exosomes secreted by cells of its murine host. Ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) analysis reveals a 9- to 62-fold enrichment of plasmalogens, as well as other classes of ether glycerophospholipids, along with a relative lack of cholesterol and sphingomyelin (SM) in the nematode exosomes compared with those secreted by murine cells. Biophysical analyses of the membrane dynamics of these exosomes demonstrate increased rigidity in those from the nematode, and parallel studies with synthetic vesicles support a role of plasmalogens in stabilizing the membrane structure. These results suggest that nematodes can maintain exosome membrane structure and integrity through increased plasmalogens, compensating for diminished levels of other lipids, including cholesterol and SM. This work also illuminates the prevalence of plasmalogens in some EVs, which has not been widely reported and could have implications for the biochemical or immunomodulatory properties of EVs. Further comparative analyses such as those described here will shed light on diversity in the molecular properties of EVs that enable them to function in cross-species communication.

RNA-mediated degradation of microRNAs: A widespread viral strategy?

Regulation of small RNAs by other non-coding RNAs is a ubiquitous feature of gene regulatory systems that can be exploited by viruses. Examples of this have been described in 3 different herpesviruses, where viral non-coding RNAs bind to highly abundant cellular (miRNAs), mediating their degradation: miR-27 is targeted by both murine cytomegalovirus and herpesvirus saimiri, while the miR-17 family is targeted by human cytomegalovirus. We review what is known about RNA-mediated regulation of miRNA stability and propose 3 potential roles that viral non-coding RNAs might assume to initiate the destruction of a miRNA, acting as "recruiters," "localizers" or "exposers." Whereas the miRNAs (miR-17 and miR-27) appear to be ancient and pre-date the common ancestor of all mammalian herpesviruses, comparative analyses of herpesvirus genomes indicate that the 3 known viral regulators of miRNA each evolved independently, and much more recently. Noting that the anti-viral activity of miRNAs might be countered by a variety of mechanisms, we propose that (i) there has been continual turnover of these mechanisms during herpesvirus evolution, and (ii) there may be many other, as yet undescribed, anti-miRNA activities encoded by other herpesviruses and indeed by viruses from other families.

Efficient Biodistribution and Gene Silencing in the Lung epithelium via Intravenous Liposomal Delivery of siRNA.

RNA interference (RNAi) may provide a therapeutic solution to many pulmonary epithelium diseases. However, the main barrier to the clinical use of RNAi remains the lack of efficient delivery vectors. Research has mainly concentrated on the intranasal route of delivery of short interfering RNA (siRNA) effector molecules for the treatment of respiratory diseases. However, this may be complicated in a diseased state due to the increased fluid production and tissue remodeling. Therefore, we investigated our hydration of a freeze-dried matrix (HFDM) formulated liposomes for systemic delivery to the lung epithelium. Here, we show that 45 ± 2% of epithelial murine lung cells receive siRNA delivery upon intravenous (IV) liposomal administration. Furthermore, we demonstrate that liposomal siRNA delivery resulted in targeted gene and protein knockdown throughout the lung, including lung epithelium. Taken together, this is the first description of lung epithelial delivery via cationic liposomes, and provides a proof of concept for the use of IV liposomal RNAi delivery to specifically knockdown targeted genes in the respiratory system. This approach may provide an attractive alternate therapeutic delivery strategy for the treatment of lung epithelium diseases.Molecular Therapy - Nucleic Acids (2013) 2, e96; doi:10.1038/mtna.2013.22; published online 4 June 2013.

Potent inhibition of Hendra virus infection via RNA interference and poly I:C immune activation.

Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus that causes fatal disease in a wide range of species, including humans. HeV was first described in Australia in 1994, and has continued to re-emerge with increasing frequency. HeV is of significant concern to human health due to its high mortality rate, increasing emergence, absence of vaccines and limited post exposure therapies. Here we investigate the use of RNA interference (RNAi) based therapeutics targeting HeV in conjunction with the TLR3 agonist Poly I:C and show that they are potent inhibitors of HeV infection in vitro. We found that short interfering RNAs (siRNAs) targeting the abundantly expressed N, P and M genes of HeV caused over 95% reduction of HeV virus titre, protein and mRNA. Furthermore, we found that the combination of HeV targeting siRNA and Poly I:C had an additive effect in suppressing HeV infection. Our results demonstrate for the first time that RNAi and type I interferon stimulation are effective inhibitors of HeV replication in vitro and may provide an effective therapy for this highly lethal, zoonotic pathogen.