PROTEOME-WIDE IDENTIFICATION OF RNA-BINDING PROTEINS PLAYING CRITICAL ROLES IN VIRUS INFECTION

Many viruses that infect humans have a genome made of RNA instead of DNA, including human immunodeficiency virus, hepatitis C virus and influenza virus. The viral enzymes involved in RNA replication display a high mutation rate, allowing rapid evolution of the virus, which helps it to evade immune defences and allows the emergence of resistance to antiviral drugs. RNA genomes are small, often encoding just a dozen proteins. By contrast, the human host cell dedicates ~1,500 RNA-binding proteins (RBPs) to RNA metabolism. Since viral genomes can only encode a handful of these proteins they rely on host proteins to complete their biological cycle. Host RBPs can also play another important role in infection, by acting as "sensors" that detect unusual molecular signatures present in viral RNAs and their replication intermediaries. Upon binding, these "sensors" trigger the antiviral response to alert neighbouring cells and provide an opportunity to block virus infection. Despite their relevance, the scope of RBPs involved in virus infection remains largely unknown. We propose, here, a new strategy to identify in a global manner the subset of RBPs implicated in infection using the prototypical Sindbis virus as a model. In brief, we will label viral RNA with a nucleotide analogue called 4-thiouridine (4SU). Upon irradiation, with 365 nm ultraviolet light, 4SU is activated acting as a "glue" that covalently links the viral RNA to the proteins interacting with it. These chemically "frozen" complexes will be captured using oligo(dT) beads as a "fishing net". The proteins "stuck" to the viral RNA will be identified by proteomic approaches. The levels or activity of key candidates will be altered using genetic tools or drugs to assess their consequences in the infection of Sindbis virus and other human RNA viruses. RBPs with a strong influence in infection will be studied in detail to understand their biological role. Our approach will identify targets for new antiviral strategies.

RNA viruses rely extensively on host RNA-binding proteins (RBPs) to accomplish their biological cycle, offering new possibilities of antiviral intervention. However, the repertoire of RBPs involved in virus infection has remained largely uncharacterised. To uncover the scope of RBPs that interact with viral RNAs in infected cells, we will develop a new method, referred to here as viral (v) RNP capture, using Sindbis virus (SINV) as discovery model. In brief, viral RNAs will be labelled with the nucleotide analogue 4-thiouridine (4SU) in presence of actinomycin D, which inhibits host RNA polymerases, directing 4SU incorporation exclusively to viral RNAs. After labelling, crosslinking between RBPs and viral RNAs will be achieved by irradiation with 365nm ultraviolet light. Proteins covalently linked to viral RNAs will be purified following stringent purification via oligo (dT) and identified quantitative proteomics. Resulting datasets will be used to select candidates for functional assays. Applying an in-house developed mid-throughput approach, we plan to study the effects of RBP overexpression, knock out and inhibition not only in cells infected with SINV but also with closely related and unrelated human pathogens. Finally, the role of RBPs exhibiting strong phenotypes in infection will be studied from a mechanistic perspective combining state-of-the-art methods in virology, RNA biology, super-resolution microscopy and proteomics. We expect to identify fundamental RBPs with regulatory roles in infection that can become antiviral targets for host-based therapies.

INTRODUCTION: RNA is a central molecule in virus biology and, thus, host RNA-binding proteins (RBPs) are expected to play major roles in infection. Indeed, distant viruses tend to interplay with similar scopes of pro- and anti-viral RBPs. However, the scope of RBPs involved in virus infection remains largely unknown. We propose, here, new approaches to untangle this key problem using Sindbis virus (SINV) as a discovery model. SINV infection in humans causes high fever, arthralgia, malaise and rash and has been linked to Pogosta, Ockelbo and Karelian diseases. It belongs to the Togaviridae family and Alphaviridae genus, which includes important human pathogens such as Chikungunya virus (CHIKV). Historically, SINV has been used as prototypical model of infection, sharing many molecular principles with closely related Alphaviruses and, to some extent, with more distant RNA viruses such as Flaviviruses.
BASIC SCIENCE: Our proposal is a basic science project; it addresses fundamental, yet highly relevant issues that will allow us to identify the cellular and molecular pathways involved in Alphavirus infections, with strong potential to be relevant in other infection models. We plan to pilot a new method, called viral ribonucleoprotein (vRNP) capture in SINV-infected cells, to provide a comprehensive census of RBPs interacting with the viral RNA. This data, together with complementary results available in our laboratory, will be used to identify potential pro- and antiviral cellular RBPs, whose impact will be studied in cells infected with SINV and other viruses.
IMPACT ON HUMAN HEALTH, PHARMA AND BIOTECH: Our research project will identify cellular proteins important in the regulation of virus infection. This will have a major impact in the discovery of therapeutic targets to block Alphavirus infection, with potential applicability to a broad spectrum of RNA viruses. In other words, our research proposal is likely to have a potential long-term impact on human global disease. This impact will be realised through dissemination of our research findings to industry, healthcare professionals, government and public sector bodies and charities. We are currently establishing relationships with industrial partners.
IMPACT ON WIDER PUBLIC: The project will also provide scope for public engagement having impact on better understanding and appreciation of the importance of cellular resources in virus infections and its potential to find therapeutic targets. The general public will benefit from the proposed project not only because of the potential therapeutic applications developed from our discoveries, but also because of our efforts to enhance public understanding of our research by engaging school students and participating in University activities directed to a general audience such as initiatives by the Oxford Trust, Open Days and Research Showcases.
IMPACT ON GENERATION OF SCIENTIFICALLY LITERATE WORK FORCE: One of the more immediate outcomes of the project will be the professional training of the post-doctoral researcher employed. This post-doc scientist will have an opportunity to learn and improve a wide range of techniques and transferable skills in virology, proteomics, RNA biology, cell biology and data analysis. This will equip her/him well for a career as scientist in academia or in the private sector. The highly skilled post-doc that we will produce will most certainly lead to wealth creation through the applications of this transferable skills base.
MRC STRATEGIC PRIORITIES: The proposed project fulfils a number of MRC strategic aims such as: "maintaining world-class UK Bioscience by supporting the best people and best ideas" and "providing skilled researchers needed for academic research". It also has particular relevance to MRC's strategic priorities in combating human infectious diseases both in the UK as well as in low and middle-income countries.