Alterations in fatty acid metabolism during RNA virus infections

Abstract

Fatty acid metabolism comprises a plethora of anabolic and catabolic reactions aimed at providing the cell with structural components, energetic resources and signaling elements, according to cellular needs. RNA viruses heavily depend on, and in most cases manipulate, host cell lipids and lipid metabolic pathways to coordinate and complete their life cycle, by obtaining the energy and molecular resources needed for viral replication, assembly and release. As such, fatty acid metabolism is an attractive target for novel antiviral therapies. Influenza A virus (IAV), a negative-sense single-stranded RNA (ssRNA) virus, is known to depend on lipid rafts for efficient viral envelope fusion, uncoating, virion assembly and budding, but little is known about its relationship with fatty acid metabolism. A number of genetic and proteomic screens designed to identify host factors involved in virus infections, including those performed in our laboratory revealed several lipid metabolic enzymes as prominent hits. Preliminary experiments using immunoblotting to measure differences in protein expression of transcription factors and several rate-limiting enzymes of lipid biosynthesis, revealed lower abundance of fatty acid synthase (Fasn), an essential mediator of de novo fatty acid synthesis, at late stages of IAV infection. Enzyme-based colorimetric quantification of total triacylglycerols, one of the end products of fatty acid (FA) synthesis, showed that triacylglycerol levels decrease early during infection. Inhibition of Fasn activity using C75, a small molecule inhibitor, or siRNA-mediated gene knockdown, in IAV infected cells dramatically impaired IAV replication. Furthermore, siRNA-mediated knockdown of fatty acid transporter 4 (Fatp4), an acyl-coenzyme A (CoA) synthase involved in FA import also resulted in drastically decreased viral replication. Metabolic labeling of cells infected with dengue virus (DENV), a positive-sense ssRNA virus, revealed distinct patterns of fatty acid modification of proteins (acylation) between mock- and infected cells. Further analysis of acylated proteins by mass spectrometry revealed heavier acylation of proteins related to FA metabolism and mitochondria in DENV infected cells; among the hits identified, were Fasn and Fatp4. Electron microscopy imaging showed extensive mitochondria-endoplasmic reticulum (mito-ER) contact regions in DENV infected cells, compared to mock-infected controls. Mito-ER sites are active centers of phospholipid synthesis, another class of end products of fatty acid metabolism. Differential ultracentrifugation of organelles from mock- and DENV-infected cells, followed by immunoblotting, indicated that both Fasn and Fatp4 localize to mito-ER sites upon infection. Furthermore, siRNA-mediated knockdown of either Fasn or Fatp4 inhibited DENV production - reminiscent of our observations with influenza. These results underscore a crucial requirement of Fasn and Fatp4 activity for replication efficiency of IAV and DENV, suggesting that RNA viruses in general might have similar needs. For DENV, these data support a recent model where various branches of fatty acid metabolism are recruited to viral replication complexes at the ER, to support membrane synthesis and virion production. Overall, this work supports fatty acid metabolism as a universal target of RNA viruses and offers a promising host-directed therapeutic target for RNA virus infections.