A spatial proteomic approach to identify members involved in the IFN response to HCMV

Abstract

During the early stages of viral detection, signalling and translocation by key protein mediators initiate antiviral activity. For example, interferon (IFN) signals through the JAK/STAT pathway, leading to phosphorylation of STAT1/2 and movement in complex with IRF9 into the nucleus. To identify novel proteins involved in the IFN response to viruses, we have employed a variety of unbiased, spatial proteomic screens that detect translocation of proteins between different organelles on a global scale. Using three different spatial screens in IFN-stimulated or control human foreskin fibroblasts, we quantified movement of >8000 proteins between the nucleus, cytoplasm and individual organelles. A novel method enabled simultaneous analysis of >2000 phosphoproteins. Proteins were shortlisted based on significant, reproducible movement. Results were validated by appropriate movement of STAT and IRF9 controls. To focus on proteins involved in the IFN response that are important during HCMV replication, we used a complementary screen of proteins that are rapidly downregulated or degraded by HCMV. The results of these screens will be presented, including a shortlist of novel factors that may have new roles in the IFN response to HCMV.