Immune evasion strategies of (+)RNA viruses

Lipid droplets accumulate in cells to serve as storage organelles. However, upon infection various immune complexes involved in innate immune responses are enriched on the lipid droplets. In particular, substrates that are modified by interferon stimulated gene 15 (ISG15) - a post translational modifier closely related to ubiquitin - are dramatically increased on lipid droplets when macrophages are either infected or treated with type I interferon. Many viruses encode for de-ISGylase enzymatic activities (e.g coronaviruses) or utilise a host de-ISGylase to hydrolyse ISGylated proteins and utilise lipid droplets for replication/assembly of viral progenies. This project will investigate whether lipid droplets have a direct role in cellular immune responses during virus infections via two specific questions: Aim 1: Whether lipid droplets serve as innate immune signaling platforms during virus infections. We will use a combination of mass spectrometry and biochemical/cell biological assays to characterise the proteome of lipid droplets when cells are stimulated with interferon-I or infected with wild-type or mutant flavi/coronaviruses and measure their impact on specific immune signaling cascades. Aim 2: Understand the mechanism by which viral proteases (or other viral factors) are able to subvert these responses, such as by hydrolysis of ISG15 modifications from LD-associated proteins to evade innate immune responses. To address both the questions we will use epithelial cells and monocyte-derived macrophages that serve as targets of both flavivirus and coronavirus infections. Infection will be performed with specific strains of viruses (e.g. the African and Asian lineages of Zika virus), which display distinct abilities to suppress host innate immune responses and hydrolyse lipid droplets, to characterise differences underlying this phenomenon.