The relevance of lipid droplet biogenesis to the hepatitis C virus replication organelle

Abstract

A hallmark of plus-strand RNA virus infection is the reshuffling of host cell membranes to form viral replication organelles. In the case of hepatitis C virus (HCV) infection, this replication organelle is called “membranous web” and appears as accumulation of double membrane vesicles (DMVs) that are derived from the endoplasmic reticulum (ER) membrane. Furthermore, lipid droplets (LDs) are often associated with the HCV membranous web and play an important role during the HCV morphogenesis. As such, the diacylglycerol-O-acyltransferase 1 (DGAT1) protein, which catalyzes the rate-limiting step of the triglyceride synthesis important for LD biosynthesis, is required for the assembly of HCV particles. Surprisingly, we found that the overexpression of the DGAT1 isozyme, DGAT2, strongly inhibits HCV infection. In this study, we aim to identify the viral and cellular determinants behind this inhibitory phenotype. We found that DGAT2 specifically inhibits the replication step of the HCV viral life cycle, while it had minimal effect on the replication of other RNA viruses of the Flaviviridae, Hepeviridae and Coronaviridae families. Using correlated light electron microscopy, we could correlate this phenotype to a defect in DMV formation. While this effect depended on the enzymatic activity of DGAT2, the mere LD accumulation was not sufficient to hamper HCV RNA replication. Analysis of a series of DGAT2 mutants revealed that reticular rather than LD association of DGAT2 is crucial to its antiviral activity. Since both DMVs and LDs are derived from the ER membrane, we hypothesize that the DGAT2-induced LD biosynthesis alters the ER lipid landscape and thereby impairs the DMV formation. We used a combination of lipidomics analysis, fluorescent lipid biosensor imaging and lipid supplementation assays to identify key lipid species involved in the DGAT2 sensitivity of HCV. Interestingly, various membrane lipid classes, especially phospholipids were enhanced upon HCV infection, but also DGAT2 overexpression. Thereby, especially phospholipids with highly unsaturated fatty acyl chains were stimulated in both conditions, suggesting a co-utilization of these lipids for both DMV and LD biogenesis. On the other hand, excess DGAT2 expression depleted certain lipid species, in particular oleyl-phospholipids, which might play an essential role during the HCV replication organelle formation. Altogether, our results indicate that lipid exchanges during DGAT2 mediated LD biogenesis influence not only the neutral but also membrane lipid composition, deleterious for the HCV replication organelle formation. The lipid changes observed globally in DGAT2 expressing cells might be relevant locally in specific ER subdomains, in physiological conditions. Furthermore, the increased lipid flux between ER and LD compartment upon exaggerated LD biogenesis may be important during steatohepatitis. Altogether, LD biogenesis mediated by the DGAT proteins might govern the spatial compartmentalization of HCV replication and assembly sites within the membranous web.