The interplay of fatty acid synthesis, metabolism, and immune function during Flavivirus infection

Abstract

Macrophage activation and Flavivirus infection can both fundamentally alter cellular metabolism and change the lipid landscape of cells. Flaviviruses upregulate fatty acid synthesis to build membranous replication complexes, and can upregulate mitochondrial metabolism to fuel replication. Classically-activated pro-inflammatory macrophages actively dysregulate mitochondrial respiration and increase fatty acid synthesis, import and accumulation whilst anti-inflammatory macrophages upregulate fatty acid oxidation to fuel mitochondrial respiration. This metabolic shift in macrophages defines and drives the functional phenotype. Flaviviruses have a tropism for macrophages, yet the metabolic profile of infected cells and its link to inflammation have never been investigated. Thus, we hypothesised that virus-induced alterations on the lipid landscape would stimulate and alter metabolic pathways, potentially contributing to the inflammatory state and response of macrophages. <br /> To test this hypothesis, we interrogated the role of fatty acid metabolism and mitochondrial respiration during infection of both macrophages and epithelial cells with West Nile (WNV) and Zika (ZIKV) virus. Our initial analyses focussed on chemical inhibition of fatty acid synthesis and oxidation, primarily targeting acetyl-CoA carboxylase (ACC - the rate limiting step of fatty acid synthesis), fatty acid synthase (FASN – the sole enzyme capable of de novo fatty acid synthesis) and carnitine palmitoyl transferase-1 (CPT1 – a mitochondrial enzyme integral for fatty acid oxidation). We observed that TOFA-mediated inhibition of ACC, (i.e. restricting de novo fatty acid synthesis) completely ablated the production of viral protein and infectious virus for both WNV and ZIKV, demonstrating the necessity of this pathway during infection. We observed some differential impact when we specifically inhibited different enzymatic domains of FASN using c75 and orlistat, such that orlistat demonstrated a higher degree of antiviral activity in both cell types, despite inhibiting an enzymatic activity downstream of c75. <br /> Inhibiting fatty acid import into the mitochondria with Etomoxir caused a reduction in infectious virus production but not protein production, suggesting that this pathway is required to fuel later steps in the viral life cycle. In addition, we observed limited induction or utilisation of lipid droplets over the course of WNV and ZIKV replication. <br /> Intriguingly, we also observed an unexpected lipid accumulation and mitochondrial fragmentation during orlistat treatment, which has not previously been reported. This fragmentation was associated with major perturbations in mitochondrial respiration and glycolysis. These observations indicate that the antiviral effect of orlistat was not necessarily a result of fatty acid deprivation but rather another unknown (potentially mitochondria-dependent) mechanism that warrants further investigation. <br /> Interestingly, although dysfunctional mitochondria underpin pro-inflammatory macrophage function, neither virus induced changes in mitochondrial respiration or glycolysis. Importantly, we observed that virus infection did induce macrophage polarisation as measured by upregulation of the cell surface markers CD80 and MHC-II. This polarisation occurred independently of fatty acid synthesis and oxidation, as inhibition of these pathways, even with TOFA, did not impact on macrophage status. We employed CRISPR-Cas9 to deplete human macrophages of FASN and showed again that efficient WNV and ZIKV replication critically dependent on FASN. However, despite repeated attempts we could not induce nor evaluate polarisation of these macrophages. <br /> We conclude that the critical role of fatty acid synthesis during flavivirus infection primarily contributes to intracellular virus replication that is uncoupled to cellular metabolism and immune activation. This data highlights the potential to target fatty acid synthesis for the development of effective antivirals. Additionally, although we observed the upregulation of classical macrophage activation markers, the maintenance of metabolic homeostasis that we observed during infection may be indicative of reduced downstream immune responses, which warrants further research.