Systems Virology Identifies a Mitochondrial Fatty Acid Oxidation Enzyme, Dodecenoyl Coenzyme A Delta Isomerase, Required for Hepatitis C Virus Replication and Likely Pathogenesis

Abstract

We previously employed systems biology approaches to identify the mitochondrial fatty acid oxidation enzyme dodecenoyl coenzyme A delta isomerase (DCI) as a bottleneck protein controlling host metabolic reprogramming during hepatitis C virus (HCV) infection. Here we present the results of studies confirming the importance of DCI to HCV pathogenesis. Computational models incorporating proteomic data from HCV patient liver biopsy specimens recapitulated our original predictions regarding DCI and link HCV-associated alterations in cellular metabolism and liver disease progression. HCV growth and RNA replication in hepatoma cell lines stably expressing DCI-targeting short hairpin RNA (shRNA) were abrogated, indicating that DCI is required for productive infection. Pharmacologic inhibition of fatty acid oxidation also blocked HCV replication. Production of infectious HCV was restored by overexpression of an shRNA-resistant DCI allele. These findings demonstrate the utility of systems biology approaches to gain novel insight into the biology of HCV infection and identify novel, translationally relevant therapeutic targets.Lipids play a role in numerous steps of the hepatitis C virus (HCV) replication cycle, including RNA replication associated with the lipid droplet (6, 35, 48), virus uptake, assembly, and secretion in association with cellular apolipoproteins (2,9,16,23,33,37,44), fusion with host membranes during virus entry (13), endocytic trafficking (3, 8), and reorganization of cellular membranes associated with replication and assembly (1,4,49,51). Furthermore, patients with HCV often exhibit hepatic steatosis and the upregulation of a number of genes involved in hepatic lipid metabolism (11,52).Recently, using integrated modeling efforts combining proteomic and lipidomic data, we identified two enzymes, dodecenoyl coenzyme A (CoA) delta isomerase (DCI) and hydroxyacyl-CoA dehydrogenase beta subunit (HADHB), that were upregulated during in vitro infection and in patients with histological evidence of fibrosis. These two enzymes were predicted to be bottleneck proteins, key regulators of the HCVinduced temporal alterations in cellular metabolic homeostasis during viral replication (11). DCI and HADHB are localized to the inner mitochondrion and catalyze the degradation of longchain fatty acids during fatty acid ␤-oxidation (7, 38, 47). Previously, ␤-oxidation was shown to be required for measles virus (46) and dengue virus replication (14), although DCI and HADHB were not directly implicated in this process. Several proteins involved in lipid catabolism, including lipases, esterases, acyl-CoA dehydrogenases, and palmitoyltransferases have also been identified as important host factors in HCV replication by RNA interference-based screening (5,8,31,40,45,49). DCI and HADHB were previously identified as host factors producing a subtle decrease in RNA production in a replicon system, although their function in HCV replication remains unknown (45). In this study, we evaluated the role of DCI by using short hairpin RN...

Linear and branched alkyl-esters and amides of gallic acid and other (mono-, di- and tri-) hydroxy benzoyl derivatives as promising anti-HCV inhibitors

“…Any perturbation of the lipid metabolism, for example inhibition of fatty acid oxidation, might result indirectly in the inhibition of HCV replication [36]. Candidate drugs can perturb the lipid metabolism of a cell in different ways, for example by inducing phospholipidosis and steatosis, as reviewed in Ref.…”

The Conundrum of Relapse in STAT-C Therapy: Does HCV Play the Red Queen or Rip Van Winkle?

“…In addition to inducing PAMP-driven responses, HCV replication also has been shown to place demands on cellular protein and lipid metabolism. [89][90][91] Not surprisingly, levels of HCV replication have been shown to be modulated by a growing number of cellular factors. 89,92-95 HCV particle production by liver cells is considered to reflect the level of replication of HCV in infected hepatocytes.…”

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

“…While the MPLEx protocol has been well-established for general extraction of lipids and metabolites 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,34, its comparison to common soil protein extraction methods for microbial analyses, such as soil protein extraction kits (see Table of Materials ) and SDS (sodium dodecyl sulfate) extractions 35 , is further evaluated here. To assess these techniques, Kansas native prairie soil proteins were extracted with each approach and analyzed directly with reversed-phase LC-MS/MS using a UPLC system coupled with a hybrid quadrupole/Orbitrap mass spectrometer.…”

“…This method was originally developed for total lipid extractions 9,11 and more recently was amended for the simultaneous extraction of metabolites, proteins, and lipids from a single sample 12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30, enabling less sample quantity and experimental variability 10 . In the MPLEx protocol, chloroform is not miscible with water, which provides the basis for the triphasic chemical separation of sample constituents into distinct fractions.…”