Systematic analysis of antiviral microRNA function

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).

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).

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.