MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles

Abstract

Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny. The human genome encodes more than 30 phospholipase A2s. These enzymes cleave fatty acids at the C2 atom of phosphoglycerides and thus modulate membrane properties. Among all PLA2s only PLA2G4A, which is recruited to perinuclear membranes by Ca²⁺ and activated by extracellular stimuli via the mitogen activated protein kinase pathway, specifically cleaves lipids with arachidonic acid. Metabolism of arachidonic acid yields prostaglandins and leukotriens, important lipid mediators of inflammation. We show that inhibition of PLA2G4A produces aberrant HCV particles and that infectivity is rescued by addition of arachidonic acid. Our results suggest that a specific lipid (arachidonic acid) is essential for production of highly infectious HCV progeny, likely by creating a membrane environment conducive for efficient incorporation of crucial host and viral factors into the lipid envelope of nascent particles. Strikingly, PLA2G4A is also essential for production of highly infectious Dengue Virus (DENV) particles but not for vesicular stomatitis virus (VSV). These observations argue that HCV and DENV which unlike VSV produce particles at intracellular membranes usurp a common host factor (PLA2G4A) for assembly of highly infectious progeny. These findings open new perspectives for antiviral intervention and highlight thus far unrecognized parallels in the assembly pathway of HCV and DENV.