The ability of a host to curb a viral infection is heavily reliant on the effectiveness of an initial antiviral innate immune response, resulting in the upregulation of interferon (IFN) and, subsequently, IFN-stimulated genes (ISGs). ISGs serve to mount an antiviral state within a host cell, and although the specific antiviral function of a number of ISGs has been characterized, the function of many of these ISGs remains to be determined. Recent research has uncovered a novel role for a handful of ISGs, some of them directly induced by IFN regulatory factor 3 in the absence of IFN itself. These ISGs, most with potent antiviral activity, are also able to augment varying arms of the innate immune response to viral infection, thereby strengthening this response. This new understanding of the role of ISGs may, in turn, help the recent advancement of novel therapeutics aiming to augment innate signaling pathways in an attempt to control viral infection and pathogenesis.

Lipid droplets (LDs) are increasingly recognized as critical organelles in signalling events, transient protein sequestration and inter-organelle interactions. However, the role LDs play in antiviral innate immune pathways remains unknown. Here we demonstrate that induction of LDs occurs as early as 2 h post-viral infection, is transient and returns to basal levels by 72 h. This phenomenon occurs following viral infections, both in vitro and in vivo. Virally driven in vitro LD induction is type-I interferon (IFN) independent, and dependent on Epidermal Growth Factor Receptor (EGFR) engagement, offering an alternate mechanism of LD induction in comparison to our traditional understanding of their biogenesis. Additionally, LD induction corresponds with enhanced cellular type-I and -III IFN production in infected cells, with enhanced LD accumulation decreasing viral replication of both Herpes Simplex virus 1 (HSV-1) and Zika virus (ZIKV). Here, we demonstrate, that LDs play vital roles in facilitating the magnitude of the early antiviral immune response specifically through the enhanced modulation of IFN following viral infection, and control of viral replication. By identifying LDs as a critical signalling organelle, this data represents a paradigm shift in our understanding of the molecular mechanisms which coordinate an effective antiviral response.

Lipid droplets (LDs) contribute to key pathways important for the physiology and pathophysiology of cells. In a homeostatic view, LDs regulate the storage of neutral lipids, protein sequestration, removal of toxic lipids and cellular communication; however, recent advancements in the field show these organelles as essential for various cellular stress response mechanisms, including inflammation and immunity, with LDs acting as hubs that integrate metabolic and inflammatory processes. The accumulation of LDs has become a hallmark of infection, and is often thought to be virally driven; however, recent evidence is pointing to a role for the upregulation of LDs in the production of a successful immune response to viral infection. The fatty acids housed in LDs are also gaining interest due to the role that these lipid species play during viral infection, and their link to the synthesis of bioactive lipid mediators that have been found to have a very complex role in viral infection. This review explores the role of LDs and their subsequent lipid mediators during viral infections and poses a paradigm shift in thinking in the field, whereby LDs may play pivotal roles in protecting the host against viral infection.

The cellular localisation of many innate signalling events following viral infection has yet to be elucidated, however there has been a few cases in which membranes of certain cellular organelles have acted as platforms to these events. Of these, lipid droplets (LDs) have recently been identified as signalling platforms for innate TLR7 and 9 signalling. Despite their wide range of similar roles in various metabolic pathways, LDs have been overlooked as potential platforms for antiviral innate signalling events. This study established an in vitro model to evaluate the efficiency of the early innate immune response in cells with reduced LD content to the viral mimics, dsDNA and dsRNA, and Sendai viral infection. Using RT-qPCR, the expression of IFN-β and IFN-λ was quantified following stimulation along with the expression of specific ISGs. Luciferase based assays evaluated the combined expression of ISRE-promoter driven ISGs under IFN-β stimulation. Cellular LD content did not alter the entry of fluorescently labelled viral mimics into cells, but significantly decreased the ability of both Huh-7 and HeLa cells to produce type I and III IFN, as well as downstream ISG expression, indicative of an impeded innate immune response. This observation was also seen during Sendai virus infection of HeLa cells, where both control and LD reduced cells replicated the virus to the same level, but a significantly impaired type I and III IFN response was observed in the LD reduced cells. In addition to altered IFN production, cells with reduced LD content exhibited decreased expression of specific antiviral ISGs: Viperin, IFIT-1 and OAS-1 under IFN-β stimulation; However the overall induction of the ISRE-promoter was not effected. This study implicates a role for LDs in an efficient early innate host response to viral infection and future work will endeavour to determine the precise role these important organelles play in induction of an antiviral response.

27Lipid droplets (LDs) are increasingly recognized as critical organelles in signalling events, 28 transient protein sequestration and inter-organelle interactions. However, the role LDs play in 29 antiviral innate immune pathways remains unknown. Here we demonstrate that induction of 30 LDs occurs as early as 2 hours post viral infection, is transient, and returns to basal levels by 31 72 hours. This phenomenon occurred following viral infections, both in vitro and in vivo. 32 Virally driven LD induction was type-I interferon (IFN) independent, however, was 33 dependent on EGFR engagement, offering an alternate mechanism of LD induction in 34 comparison to our traditional understanding of their biogenesis. Additionally, LD induction 35 corresponded with enhanced cellular type-I and -III IFN production in infected cells, with 36 enhanced LD accumulation decreasing viral replication of both HSV-1 and Zika virus 37 (ZIKV). Here, we demonstrate for the first time, that LDs play vital roles in facilitating the 38 magnitude of the early antiviral immune response specifically through the enhanced 39 modulation of IFN following viral infection, and control of viral replication. By identifying 40 LDs as a critical signalling organelle, this data represents a paradigm shift in our 41 understanding of the molecular mechanisms which coordinate an effective antiviral response. 42 48The role of LDs in an infection setting has not been well studied, however, it has been 49 demonstrated that LDs accumulate in leukocytes during inflammatory processes, and they are 50 also induced in human macrophages during bacterial infections 2 . Multiple bacterial strains, 51 including Mycobacterium spp., Chlamydia spp., Klebsiella spp. and Staphylococcus spp. are 52 known to upregulate LDs very early following bacterial infection in both primary and cell 53 culture macrophage models, and this has also been seen for a number of bacterial species in 54 rodent macrophage cell lines 5-7 . Interestingly, Trypanosoma cruzi infection of macrophages 55 also induces LDs, however, this response takes 6-12 days to occur following infection 8 . 56Bacterial-induced LD induction in immune cells has been shown to depend on toll-like 57 receptor engagement, mainly via TLR2 and TLR4, however, the role of LDs in the outcome 58 of bacterial infection remains largely unknown, and the exact mechanisms for controlling LD 59 induction remain elusive 9,10 . It has been suggested in recent work in the zebrafish model that 60 embryos with higher levels of LDs are more protected from bacterial infections 11 and work 61 in the Drosophila embryo has demonstrated that LDs can bind to histones which are released 62 upon detection of intracellular bacterial LPS and act in a bactericidal manner 12 . 63 Interestingly, LD induction has been demonstrated to be a direct result of immune activation 64 of macrophages by IFN-ߛ in a HIF-1 dependent signalling pathway 13 . M. tuberculosis 65 acquires host lipids in the absence of LDs under normal conditions, however, IFN-ߛ ...