CHARACTERISATION OF A NOVEL ROLE FOR PKR IN INHIBITING VIRUS ASSEMBLY (360G-Wellcome-302268_Z_23_Z)

The interferon-induced, double-stranded RNA-activated kinase (PKR) is a key component of the innate antiviral response. We seek to understand a novel function of PKR revealed by recent work on the hepatitis C virus (HCV) NS5A protein: both virus assembly and effects of HCV on lipid droplet morphology were blocked by NS5A mutations. Intriguingly, these phenotypes were restored in cells that were silenced for expression of either PKR, or its downstream effector, the transcription factor IRF1, implicating a novel role for PKR/IRF1 in blocking virus assembly and lipid droplet morphology. We will firstly use transcriptomics to identify genes that are regulated by PKR/IRF1 in uninfected or HCV-infected cells. Candidate genes will be overexpressed and/or silenced to investigate effects on virus assembly. Secondly, we will ask whether the block to assembly by PKR/IRF1 is a pan-viral response, investigating whether over-expression of the candidate genes regulates assembly of other lipid droplet-dependent viruses. Thirdly, we will determine the mechanisms by which virus assembly is blocked: specific functions of the candidate genes (eg enzymatic activities) will be ablated by mutagenic approaches. Effects on lipid droplet composition will be investigated by lipidomics and proteomics, and virion characteristics will be investigated by biochemical and biophysical approaches.