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.

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.

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.