Proteomic approaches to elucidate metabolic changes during virus infections

Abstract

Post-translational modifications are central to spatial and temporal regulation of protein function. Ubiquitylation has emerged as a widely utilized protein modification strategy that participates in a multitude of cellular processes and often targeted by pathogens. We implemented a chemical proteomics based approach using a non-hydrolyzable analog of ubiquitin to identify substrates that are specifically modified in the context of flavivirus infections. Amongst those isolated, we identified several that belong to innate immune response pathways, lipid droplets and the ER-associated degradation machinery, and were differentially modified in an infection-dependent manner. We characterized the function of Aup1 – a lipid droplet associated type-III membrane protein in the process of flavivirus biogenesis. Aup1 is mono-ubiquitylated in normal physiology but appeared in the unconjugated form in infected cells. To understand its functional relevance we generated Aup1-deficient cells through the CRISPR/Cas9 strategy. Aup1-/- cells were resistant to virus production whereas reconstitution of wild-type Aup1 rescued this phenotype. Virus infection also resulted in a subcellular relocalization of Aup1 from lipid droplets to autophagosomes as visualized by confocal imaging and proximity based labeling. Unmodified Aup1 resulted in smaller and a more dispersed morphology of lipid droplets, promoting their rapid hydrolysis through autophagy. Our current data indicate that virus-triggered autophagy is a critical phenomenon that presents a fine balance between virus replication and host defense and ultimately determines the outcome of infection.