Lipid Droplets (LDs) were initially considered simply as a cellular energy source but are now recognised as critical organelles in signalling events, transient protein sequestration and inter-organelle interactions. Recently, our lab has demonstrated that LDs are upregulated during viral infection, and that this upregulation contributes to an enhanced interferon response from the infected cell indicating for the first time that the LD contributes to an effective immune response, however the mechanism of this is unknown.
Here, we describe for the first time that there are several critical key antiviral signalling molecules that localise to the LD during this response. We have optimised techniques to isolate pure lipid droplets from primary immortalised astrocyte cells before and following activation of viral RNA signalling pathways. Proteomic analysis has revealed there was 92 significantly upregulated proteins on LDs following stimulation with 13% of the significantly enriched proteins being associated with the interferon response. Of these, MX1 and ISG15 were significantly upregulated on LD fractions at both 8 and 24hrs following RNA viral mimic stimulation, whereas we see proteins such as viperin, RIG-I and STAT1 exclusively upregulated at 24hrs. Many significantly upregulated proteins are thought to be cytoplasmic, therefore, to confirm the localisation of these signalling proteins to the LD, a technique was designed to perform fluorescent confocal microscopy on isolated fluorescently stained lipid droplets probing for the identified immune proteins; and this, along with western blotting, has confirmed the localisation of these proteins to LDs.
Here, we demonstrate that there are important antiviral immune signalling proteins that localise to the LD following viral mimic stimulation, indicating that the LD can act as a signalling platform for signalosome formation to aid host immunity. The mechanism by which these proteins localise to the LD and the function of this is still being explored by our laboratory.