Systematic analysis of antiviral microRNA function
Lead Research Organisation:
University of Edinburgh
Department Name: Sch of Biological Sciences
Abstract
In this proposal we examine how specific molecules, microRNAs, limit the capacity of viruses to replicate in host cells. Viruses (by definition) require host proteins to enter a cell, replicate and spread in an animal; microRNAs regulate the expression level of these proteins. We have previously demonstrated that certain host microRNAs suppress replication of multiple herpesviruses (cytomegalovirus, herpes simples virus-1 and mouse gammaherpesvirus) as well as an unrelated single stranded RNA virus, Semlikiforest virus. We hypothesize that the antiviral properties of these microRNAs are based on down-regulating specific cellular proteins that different viruses rely on. A broad, long-term goal in this work is to better understand how the microRNA mechanism (subtle down regulation of multiple host proteins simultaneously) could be used to treat infection. The advantage of this type of host-targeted therapy is that it could potentially be used to treat a range of infections and reduce the likelihood that viruses could readily mutate to become resistant. Here we implement cutting-edge biochemical techniques to identify the targets of antiviral microRNAs and determine how the targets suppress viral replication. Using murine and human cytomegalovirus as model systems, we examine targets of the miRNAs that are present in both mouse and human cells (and likely other animal hosts). In parallel, we examine whether cytomegaloviruses have evolved mechanisms for blocking host microRNA function. The end goal is to determine the mechanism of action of specific antiviral microRNAs and shed light on factors that will dictate the approaches required to use these molecules in a therapeutic context (e.g. combinatorial regulation of genes by multiple microRNAs and scope for viral interference and resistance).
Technical Summary
The aim of this work is to develop the systems-level methodologies required to understand how specific host microRNAs suppress viral replication in animals and determine whether viruses have evolved mechanisms to interfere with this. We will implement biochemical approaches developed in Prof. Tollervey's lab to map precise binding sites between microRNAs and their targets within the RNA-induced silencing complex (RISC). Specifically, we propose to purify and sequence cDNAs derived from RNAs that are UV crosslinked to the Argonaute 2 (Ago) protein in infected cells (the Ago protein binds to microRNA-mRNA complexes within RISC). The data generated will be relevant to a range of research questions; here we focus on identifying host and viral gene targets of 6 microRNAs derived from two clusters, miR-199a/214 and miRNA-23/24/27, which inhibit herpesviral replication. We will use pathway analysis to build a model for how the targets of these microRNAs impact the host cell and how this suppresses cytomegalovirus replication. This model will then be tested by knocking down individual or pooled targets and measuring the effect on viral replication and host signalling using reporter assays. In parallel we will identify host genes that are subject to regulation by multiple microRNAs within a cluster and test the additive, cooperative or antagonizing effects of combinatorial regulation. Finally we will determine whether viral elements also interact with these microRNAs and use bacterial artificial chromosome technology to test the functions of these viral elements in murine cytomegalovirus infection in vitro and in vivo. The end goal is to determine the mechanism of action of specific antiviral microRNA clusters and shed light on factors that will dictate the approaches required to use these molecules in a therapeutic context (e.g. combinatorial regulation of genes by multiple microRNAs and scope for viral interference and resistance).
Planned Impact
The work detailed in this proposal is aimed at advancing the basic scientific understanding of how microRNAs impact biological processes. The primary beneficiaries will be other researchers in the microRNA field, which spans multiple areas of biology and medicine and includes academic institutions as well as industry. The results will be disseminated to other academics and industry at national and international conferences and will also be made freely available online when appropriate. The microRNA field is timely and lucrative. Support for this research in the UK is relevant to both social and economic welfare and international standing. Training research scientists in the field of miRNA research is fundamental to continued UK contribution to this research. The research questions addressed here focus on advancing our understanding of how viruses interact with host cells and how we can manipulate the cellular environment to treat infection. The worldwide antiviral market is estimated to grow from ~$18 million to as much as $25 billion by 2011 (1). New strategies are required to understand how to treat infection in a way that will not lead to resistance and will prepare the population for newly emerging viral strains. Identification of host-targeted therapeutic strategies holds commercial and social value. The importance of results obtained will be conveyed to the public through the publication of work in academic journals as well as through the University's press office, which interfaces with local and international press agencies. 1. McDCarthy, B. 2007. Antivirals-an increasingly healthy investment. Nature Biotech 25, 1390-1393.
Publications
Libri V
(2012)
Murine cytomegalovirus encodes a miR-27 inhibitor disguised as a target.
in Proceedings of the National Academy of Sciences of the United States of America
Libri V
(2013)
Regulation of microRNA biogenesis and turnover by animals and their viruses.
in Cellular and molecular life sciences : CMLS
McCaskill J
(2015)
RNA-mediated degradation of microRNAs: A widespread viral strategy?
in RNA biology
McCaskill JL
(2017)
Broad-Spectrum Inhibition of Respiratory Virus Infection by MicroRNA Mimics Targeting p38 MAPK Signaling.
in Molecular therapy. Nucleic acids
Travis AJ
(2014)
Hyb: a bioinformatics pipeline for the analysis of CLASH (crosslinking, ligation and sequencing of hybrids) data.
in Methods (San Diego, Calif.)
Description | In this work we discovered novel targets for host microRNAs that influence the ability of cytomegalovirus, and likely other viruses, to replicate. In particular we identified members of the Map Kinase signaling pathway as targets of microRNA-27, highlighting specific proteins and host cell pathways required for viral replication. We went on to identify additional host microRNAs that target the Map Kinase signaling pathway (miR-24, miR-744, miR-124) and have broad spectrum antiviral properties against a wide range of virues, including respiratory viruses. In particular we identified MK2 as a key protein in respiratory syncytial virus and influenza virus infection, whose suppression by these microRNAs reduces the ability of the viruses to replicate. This led to a new collaboration with miRagen to test delivery of these antiviral microRNA mimics to epithelial cells. This has now extended into a collaboration with Johnson & Johnson Innovations/Janssen Pharmaceuticals to identify the target networks of antiviral miRNAs and a collaboration with Hong Kong University to test the miRNA antiviral candidates in coronaviruses. During the course of this grant we also determined that for immunoprecipitation experiments to identify microRNA targets it is necessary to carry out studies with physiological concentrations of the Argonaute proteins. We therefore obtained further funding to create a genetically modified mouse with the affinity tags introduced to the endogenous Argonaute protein. |
Exploitation Route | Many investigators require the methodology that we are developing for analysis of microRNA targets in normal and disease biology. Furthermore many of the microRNA targets that we identify are relevant to understanding pathways that viruses use to establish infections in host cells and this informs new strategies for developing effective antivirals that are relevant to a range of infection models, including coronaviruses. |
Sectors | Aerospace, Defence and Marine,Healthcare |
Description | This research focuses on understanding how a type of host molecule, microRNAs, impacts viral replication. The basic research that was carried out in this project therefore informs new therapeutic strategies against viruses. Because the molecules we study operate on the host cell, rather than directly targeting the virus, this work is particularly important to human health in the age of increasing resistance to existing antivirals. As part of this work, we have also generated further tools that other investigators can use to study this class of host molecule, which play a range of roles in animal and human disease. This work is of interest to companies developing new antivirals against respiratory viruses. |
First Year Of Impact | 2013 |
Sector | Aerospace, Defence and Marine,Healthcare,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal |
Description | Institutional Strategic Support Award, Novel lung delivery of host-targeted microRNAs for the treatment of respiratory viral infections |
Amount | £35,135 (GBP) |
Organisation | University of Edinburgh |
Sector | Academic/University |
Country | United Kingdom |
Start | 11/2015 |
End | 01/2016 |
Description | Proof of Concept |
Amount | £790,000 (GBP) |
Organisation | Scottish Enterprise |
Sector | Public |
Country | United Kingdom |
Start | 04/2013 |
End | 06/2015 |
Description | Wellcome Trust Enhancement Award |
Amount | £110,142 (GBP) |
Funding ID | 097394/Z/11/A |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2015 |
End | 08/2016 |
Title | Refined methodology for identifying viral-host RNA-RNA interactions |
Description | We have refined a method for identifying direct viral-host RNA-RNA interactions that is relevant to finding new drug targets in respiratory virus infection. |
Type Of Material | Technology assay or reagent |
Year Produced | 2019 |
Provided To Others? | No |
Impact | At present the methods enable us to identify and test new antiviral strategies based on the coordinated regulation of host networks. |
Description | MicroRNA function in respiratory infections |
Organisation | Johnson & Johnson |
Department | Janssen Pharmaceutica |
Country | Global |
Sector | Private |
PI Contribution | We have developed methods to profile small RNA abundance and activity changes during infection and to directly identify the targets of microRNAs. |
Collaborator Contribution | Janssen has datasets on the functional impact of small RNAs and target RNAs during respiratory viral infection and the collaboration is based on combining or knowledge and know-how to identify small RNA-target interactions that influence viral replication and may represent new drug targets. |
Impact | None yet |
Start Year | 2018 |