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When a host cell is infected by a virus, it activates the innate immune response, setting off a cascade of signalling events leading to the production of an antiviral response. This immune response is typically robust and in general works well to clear viral infections, however, viruses have evolved evasion strategies to combat this, and therefore, a better understanding of how this response works in more detail is needed for the development of novel and effective therapeutics. Lipid droplets (LDs) are intracellular organelles and have historically been thought of simply as cellular energy sources, however, have more recently been recognised as critical organelles in signalling events. Importantly, many viruses are known to take over host cellular production of LDs, and it has traditionally been assumed the sole purpose of this is to supply energy for viral life cycle events. However, our recent work positions LDs as important organelles during the first few hours of an antiviral response, showing that they underpin the production of important antiviral cytokines following viral infection. Following infection of cells with either RNA viruses (Zika, Dengue, Influenza A) or a DNA (Herpes Simplex Virus-1) virus, LDs were rapidly upregulated, and this response was also replicated following stimulation with viral mimic agonists. This upregulation of LDs following infection was transient, and interestingly, did not follow the well described homeostatic mechanism of LD upregulation, instead being controlled by EGFR. The cell’s ability to mount an effective immune response was greatly diminished when inhibiting EGFR, thus inhibiting LD upregulation during infection, also leading to an increase in viral replication. In this microreview, we extrapolate our recent findings and discuss LDs as an important organelle in the innate immune response.

The role of interferon and interferon stimulated genes (ISG) in limiting bacterial infection is controversial, and the role of individual ISGs in the control of the bacterial life-cycle is limited. Viperin, is a broad acting anti-viral ISGs, which restricts multiple viral pathogens with diverse mechanisms. Viperin is upregulated early in some bacterial infections, and using the intracellular bacterial pathogen, S. flexneri, we have shown for the first time that viperin inhibits the intracellular bacterial life cycle. S. flexneri replication in cultured cells induced a predominantly type I interferon response, with an early increase in viperin expression. Ectopic expression of viperin limited S. flexneri cellular numbers by as much as 80% at 5hrs post invasion, with similar results also obtained for the intracellular pathogen, Listeria monocytogenes. Analysis of viperins functional domains required for anti-bacterial activity revealed the importance of both viperin’s N-terminal, and its radical SAM enzymatic function. Live imaging of S. flexneri revealed impeded entry into viperin expressing cells, which corresponded to a loss of cellular cholesterol. This data further defines viperin’s multi-functional role, to include the ability to limit intracellular bacteria; and highlights the role of ISGs and the type I IFN response in the control of bacterial pathogens.

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) are recognized as dynamic organelles and scaffolding platforms to regulate signalling cascades. Here, Monson et al. provide evidence that LDs are involved in regulation of an early antiviral immune response specifically through the enhanced modulation of IFN following viral infection in vitro and in vivo.

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Viperin is an interferon-inducible protein that is pivotal for eliciting an effective immune response against an array of diverse viral pathogens. Here we describe a mechanism of viperin's broad antiviral activity by demonstrating the protein's ability to synergistically enhance the innate immune dsDNA signaling pathway to limit viral infection. Viperin co-localized with the key signaling molecules of the innate immune dsDNA sensing pathway, STING and TBK1; binding directly to STING and inducing enhanced K63-linked polyubiquitination of TBK1. Subsequent analysis identified viperin's necessity to bind the cytosolic iron-sulfur assembly component 2A, to prolong its enhancement of the type-I interferon response to aberrant dsDNA. Here we show that viperin facilitates the formation of a signaling enhanceosome, to coordinate efficient signal transduction following activation of the dsDNA signaling pathway, which results in an enhanced antiviral state. We also provide evidence for viperin's radical SAM enzymatic activity to self-limit its immunomodulatory functions. These data further define viperin's role as a positive regulator of innate immune signaling, offering a mechanism of viperin's broad antiviral capacity.

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., Lipid droplets are key organelles in multiple cellular processes; recent advances in the field have demonstrated that they together with their lipid mediators may play pivotal roles in the outcome of viral infection.

SummaryLipid 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 hours post viral infection, is transient, and returns to basal levels by 72 hours. This phenomenon occurred following viral infections, both in vitro and in vivo. Virally driven LD induction was type-I interferon (IFN) independent, however, was dependent on EGFR engagement, offering an alternate mechanism of LD induction in comparison to our traditional understanding of their biogenesis. Additionally, LD induction corresponded with enhanced cellular type-I and -III IFN production in infected cells, with enhanced LD accumulation decreasing viral replication of both HSV-1 and Zika virus (ZIKV). Here, we demonstrate for the first time, 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.

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) have traditionally been thought of as solely lipid storage compartments for cells, however, in the last decade, they have emerged as critical organelles in health and disease. LDs are highly dynamic within cells, and their movement is critical in organelle–organelle interactions. Their dynamics are known to change during cellular stress or nutrient deprivation, however, their movement during pathogen infections, especially at very early timepoints, is under-researched. This study aimed to track LD dynamics in vitro, in an astrocytic model of infection. Cells were either stimulated with a dsRNA viral mimic, poly I:C, or infected with the RNA virus, Zika virus. Individual LDs within infected cells were analysed to determine displacement and speed, and average LD characteristics for multiple individual cells calculated. Both LD displacement and mean speed were significantly enhanced in stimulated cells over a time course of infection with an increase seen as early as 2 h post-infection. With the emerging role for LDs during innate host responses, understanding their dynamics is critical to elucidate how these organelles influence the outcome of viral infection.

Viperin is an interferon-inducible protein that is pivotal for eliciting an effective immune response against an array of diverse viral pathogens. Here we describe a mechanism of viperin’s broad antiviral activity by demonstrating the protein’s ability to synergistically enhance the innate immune dsDNA signalling pathway to limit viral infection. Viperin co-localised with the key signalling molecules of the innate immune dsDNA sensing pathway, STING and TBK1; binding directly to STING and inducing enhanced K63-linked polyubiquitination of TBK1. Subsequent analysis identified viperin’s necessity to bind the cytosolic iron-sulphur assembly component 2A, to prolong its enhancement of the type-I interferon response to aberrant dsDNA. Here we show that viperin facilitates the formation of a signalling enhanceosome, to coordinate efficient signal transduction following activation of the dsDNA signalling pathway; which results in an enhanced antiviral state. We also provide evidence for viperin’s radical SAM enzymatic activity to self-limit its immunomodulatory functions. This data further defines viperin’s role as a positive regulator of innate immune signalling, offering a mechanism of viperin’s broad antiviral capacity.

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.