We used a combination of gene silencing and pharmacologic approaches to validate our previous computational modeling predictions that dodecenoyl coenzyme A (CoA) delta isomerase (DCI)-mediated mitochondrial fatty acid oxidation plays a critical role in hepatitis C virus (HCV) replication. Both approaches confirmed the importance of DCI to the HCV replication cycle, demonstrating the value of systems biology as a means of gaining insights into HCV pathogenesis that have been overlooked by conventional approaches. DCI knockdown completely blocked viral RNA production, indicating that its mechanism of action in augmenting HCV replication occurs at or before assembly of RNA replication complexes and initiation of viral transcription by the HCV RNA-dependent RNA polymerase (RdRp). Because of DCI's essential role in the catabolism of long-chain fatty acids and its initial identification by a model built using proteomic and lipidomic data, we propose that DCI exerts its effects on HCV replication by modulating lipid content in the cell.
Alterations in lipid metabolism have long been observed in both experimental and clinical HCV infection. Specific lipid species undergo changes in composition and abundance that facilitate molecular interactions required for HCV infection. Patients with chronic HCV develop steatosis and alterations in serum lipid levels, particularly as disease progresses. Though DCI has previously not been implicated in mediating these changes, mitochondrial β-oxidation could be involved in a number of cellular processes required for HCV replication. Numerous studies have observed changes in the lipidome of infected cells corresponding with known interactions between replicating HCV and multiple host lipids or lipid-related machinery. Lipid rafts rich in sphingomyelin are required for the assembly of RNA replication complexes and activity of the HCV RdRp (1
). Lipid droplets are specialized organelles designed for the storage of neutral lipids, which attach to HCV core protein and facilitate assembly of viral replication complexes and viral particles (35
). These lipid droplets are attached to membranes where RNA replication complexes assemble. Both the induction of lipid droplet formation and the substantial organelle and membrane remodeling required for RNA replication and virion assembly at these sites necessitate substantial reprogramming of cellular lipid biosynthetic and metabolic pathways, all of which may be mediated by DCI. However, despite the wealth of information concerning the role of lipids in HCV replication and the major role of fatty acid oxidation in both meeting the cellular energetic requirements and generating building blocks for new lipid species in lipogenesis, conventional approaches have never implicated this pathway in playing an essential role in HCV replication. Without employing a systems-level approach, fatty acid oxidation would not have been identified as a key pathway regulating metabolic reprogramming required for HCV replication and pathogenesis.
Activating fatty acid oxidation pathways seems contrary to numerous reports that lipogenesis, including fatty acid synthesis, is required at virtually all stages of HCV replication. DCI may be required to break down more-complex lipid species to favor synthesis of other lipid species facilitating HCV infection. Some of these lipid species might be membrane phospholipids, such as phosphatidylinositol-4-phosphate, which nucleates assembly of RNA replication complexes on membrane surfaces (17
). Additionally, lipidation of host and viral proteins is known to play an essential role in HCV infection. For example, geranylgeranylation of host proteins is required for HCV RNA replication (24
), and the viral core and NS4B proteins are palmitoylated to facilitate interactions with membranes and other proteins required to assemble functional replication complexes (32
). By mediating the degradation of lipids not required for HCV replication, DCI generates energy and materials for the biosynthesis of other lipid molecules required during the viral life cycle.
Another possibility is that DCI specifically reduces the quantity of polyunsaturated fatty acids (PUFA) in the cell, creating a favorable environment for HCV replication. DCI specifically isomerizes mono- and polyunsaturated fatty acids with cis
double bonds at odd-numbered carbon atoms into their 2-trans
-enoyl-CoA forms (20
). HCV RNA replication is inhibited specifically by accumulation of polyunsaturated fatty acids normally degraded by DCI (30
). A characteristic feature of fasting DCI knockout mice is hepatic accumulation of unsaturated long-chain fatty acids (21
). DCI deficiency results in the accumulation of PUFA in Huh7/DCI-1 cells, subsequently inhibiting HCV replication, possibly by altering lipid rafts or membrane structure sufficiently as to prevent the assembly and function of RNA replication complexes.
Our metabolomic data provide insight into the molecular basis for HCV inhibition in DCI-deficient Huh7 cells. Consistent with DCI's function in channeling unsaturated fatty acids into the mitochondrial β-oxidation pathway, DCI knockdown results in an increase in abundance of the monounsaturated DCI substrate oleic acid. Exogenous oleic acid enhances RNA synthesis by a full-length HCV genotype 1b (HCV1b) replicon (24
), although the molecular mechanism for this enhancement and the impact of DCI-mediated catabolism during oleic acid supplementation remain undetermined. Fatty acid oxidation may exert pleiotropic effects on the HCV life cycle, including potentially significant roles in enhanced energy production and modulating the cellular composition of lipid species with pro- or antiviral effects. Either one of these potentially significant roles could influence the global metabolic reprogramming known to occur during HCV infection (11
). During conditions of impaired lipid catabolism, these effects would presumably be abrogated, forcing the cell to rely on alternative energy sources. Indeed, the observed aqueous metabolic profile further revealed that Huh7/DCI-1 cells exhibited increases in numerous amino acids and exemplary intermediates of urea synthesis. These metabolic alterations are consistent with those observed in DCI knockout mice (21
) and suggest an increased dependence on amino acids for energy production.
Alternatively, increasing evidence indicates that fatty acid remodeling substantially impacts the composition, integrity, and function of biological membranes (22
). For example, the incorporation of PUFA, and to a lesser extent oleic acid, into phosphatidylethanolamine (PE) was recently shown to modify the biophysical organization and fluidity of lipid rafts (43
). This may be due to unfavorable interactions between the disordered acyl chain of PE with sphingomyelin, resulting in PE-rich nonraft microdomains that can impact protein conformation (22
). Such membrane reorganizations adversely impact the localization of molecules that need to be clustered in close physical proximity, such as immunological synapse proteins required for antigen presentation and T cell activation (27
). The relative contribution of DCI deficiency on the composition of lipid species comprising the various structural entities supporting HCV replication remains an important unanswered question. We are actively addressing this question with additional metabolomic studies to better understand both the full breadth of the cellular lipidome in Huh7/DCI-1 cells as well as the influence of DCI deficiency on the metabolome in the context of HCV infection.
As the phenotypic impact of impaired lipid catabolism appears to be most prominent under conditions of metabolic challenge, such as dietary fasting, it will be crucial to perform additional studies aimed at characterizing global host, gene, protein, and metabolite changes occurring in DCI-deficient cells during the cellular stress induced during HCV infection. Comparative analyses of the molecular changes occurring in purified subcellular structures, including lipid rafts, lipid droplets, viral RNA replication complexes, mitochondria, and mitochondrion-associated membranes (MAM) during HCV infection of wild-type versus DCI-deficient Huh7 cells will help to refine our understanding of how metabolic reprogramming influences current models of HCV infection and pathogenesis.
Further investigation of the importance of fatty acid oxidation in vivo may lead to the development of novel HCV therapeutics targeting host factors rather than viral proteins. The HCV therapies now in development primarily target viral enzymes such as proteases and polymerases and must be used in combination with PEGylated alpha interferon and ribavirin therapy. These regimens are not tolerated well by patients and can select for drug-resistant viral mutants. Targeting host factors such as DCI is an approach that could potentially eliminate the need for multidrug regimens with severe side effects in favor of a treatment that does not select for drug-resistant viruses, may be used as monotherapy, and completely blocks HCV replication. Together, these studies demonstrate our use of systems biology to identify and evaluate a novel host factor that both advances our understanding of the biology of HCV infection and provides an attractive potential new target for treating this highly prevalent and deadly disease.