Promotion of Hendra virus replication by microRNA 146a

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease

A Functional Genomics Approach to Henipavirus Research: The Role of Nuclear Proteins, MicroRNAs and Immune Regulators in Infection and Disease.

Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are zoonotic RNA viruses that cause lethal disease in humans and are designated as Biosafety Level 4 (BSL4) agents. Moreover, henipaviruses belong to the same group of viruses that cause disease more commonly in humans such as measles, mumps and respiratory syncytial virus. Due to the relatively recent emergence of the henipaviruses and the practical constraints of performing functional genomics studies at high levels of containment, our understanding of the henipavirus infection cycle is incomplete. In this chapter we describe recent loss-of-function (i.e. RNAi) functional genomics screens that shed light on the henipavirus-host interface at a genome-wide level. Further to this, we cross-reference RNAi results with studies probing host proteins targeted by henipavirus proteins, such as nuclear proteins and immune modulators. These functional genomics studies join a growing body of evidence demonstrating that nuclear and nucleolar host proteins play a crucial role in henipavirus infection. Furthermore these studies will underpin future efforts to define the role of nucleolar host-virus interactions in infection and disease

IFN regulatory factor 8 is a key constitutive determinant of the morphological and molecular properties of microglia in the CNS.

IFN regulatory factor (IRF) 8 is a transcription factor that has a key role in the cellular response to IFN-γ and is pivotal in myeloid cell differentiation. Whether IRF8 plays a role in the development and function of microglia, the tissue-resident myeloid cells of the brain, is unknown. Here, we show IRF8 is a constitutively produced nuclear factor in microglia, which suggested that IRF8 might also be a key homeostatic transcriptional determinant of the microglial cell phenotype. In support of this, in mice with a targeted disruption of the IRF8 gene, microglia were increased in number and showed gross alterations in morphology and surface area. In situ analysis of some key myeloid markers revealed that IRF8-deficient microglia had significantly reduced levels of Iba1, but increased levels of CD206 (mannose receptor) and F4/80 as well as increased tomato lectin binding. Analysis of microglia ex vivo revealed IRF8-deficient microglia had significantly increased levels of CD45, CD11b and F4/80, but significantly decreased levels of the chemokine receptors CCR2, CCR5 and CX3CR1. The known involvement of some of these molecular markers in membrane dynamics and phagocytosis led us to examine the phagocytic capacity of cultured IRF8-deficient microglia, however, this was found to be similar to wild type microglia. We conclude IRF8 is a constitutively produced nuclear factor in resident microglia of the CNS being a crucial transcriptional determinant of the phenotype of these cells in the healthy brain

Circulating microRNA profiles of Hendra virus infection in horses

10.1038/s41598-017-06939-wScientific Reports71743

GFP⁺ cells in the CNS are exclusively CD11b⁺ microglia.

<p>Flow cytometry was performed on cells isolated from the brain of healthy adult WT (A, B) and IRF8-deficient mice as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049851#s4" target="_blank">Materials and Methods</a>. For analysis, cells were scatter-gated to exclude dead cells and GFP⁺ cells were selected (A). Greater than 99% of GFP⁺ cells expressed CD11b in WT and IRF8-deficient mice (B), consistent with a microglial phenotype. Isotype matched antibodies were used to determine background staining (data not shown).</p

The levels of some key myeloid markers are altered in the brain of IRF8-deficient mice.

<p>Immunostaining was performed on brain sections from healthy, adult WT (A–D) or IRF8-deficient (E–H) mice as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049851#s4" target="_blank">Materials and Methods</a>. Panels A, B, C, E, F, G show cortex while panels D, H show cerebellum (original magnification all panels 1000X). For immunofluorescence (G, H) DAPI was used to stain nuclei. Whole brain lysates were prepared from healthy, adult mice and 20 µg of protein analysed by western blotting (I).</p