Towards the discovery of cellular RNA-binding proteins with master regulatory roles in virus infection

RNA is a central molecule for RNA virRNA is a central molecule for RNA virus infection. However, viral genomes encode very few proteins that are able to interact with viral RNA. Hence, viruses co-opt cellular RNA-binding proteins (RBPs) to support infection. The host cell also employs RBPs to combat viruses through the recognition of unusual signatures present viral RNAs. Despite these critical roles, the complement of cellular RBPs involved in infection remains largely unknown. This research programme aims to discover comprehensively and unbiasedly the repertoire of cellular RBPs endowed with master regulatory roles in virus infection. To achieve this, we will exploit a novel method developed in my laboratory, named viral RNA interactome capture (vRIC), that allows the elucidation of viral ribonucleoprotein (RNP) composition with unprecedented depth and specificity. It employs pulse-labelling of viral RNA with a photoactivatable nucleotide analogue, UV crosslinking, viral RNA purification with antisense probes and quantitative proteomics. (Aim 1) I hypothesise that cellular RBPs that interact with a broad-range of viral RNAs are likely endowed with master regulatory roles in infection. To test this, I will apply vRIC to cells infected with different RNA viruses to discover RBPs that are shared across viral RNPs. Moreover, we will employ interferon α to investigate if the engagement of these RBPs with viral RNA is regulated by the antiviral state. (Aim 2) To test if these broad-spectrum RBPs display master regulatory roles in infection, I will apply a novel functional screen using genetically modified cells and a broad library of fluorescent viruses. (Aim 3) We will then decipher the molecular mechanisms underpinning the ability of these RBPs to support or restrict infection. In summary, this innovative research programme will discover cellular RBPs with master regulatory roles in infection with great potential as targets for broad-spectrum antiviral therapies.