Infection with Hepatitis C Virus (HCV) is a global health problem affecting ~2 percent of the world's population. In 80 percent of those afflicted infection persists and can cause severe liver damage. Fatty liver syndrome (steatosis) is a frequent condition affecting ~50 percent of chronically infected individuals. The viral core protein expressed in livers of mice recapitulates this condition and causes abnormal lipid accumulation. Core also binds lipid droplets and triggers viral assembly at the endoplasmic reticulum (ER) close to lipid droplets. Our preliminary results reveal a novel connection between both processes. We find that core exclusively induces and binds lipid droplets generated by diacylglycerol acyltransferase (DGAT) 1. In cells lacking DGAT1 or treated with a DGAT1 inhibitor, core cannot induce lipid droplets and cannot localize to lipid droplets to recruit viral RNA for encapsidation. As a consequence, HCV particle production is severely impaired. Our data support a model in which core and DGAT1 physically interact in regions of the ER that give rise to DGAT1- mediated lipid droplets loaded with core at their surface. Only these droplets serve as intracellular platforms for viral assembly in infected cells. This model bears important implications for our understanding of the HCV life cycle and the clinical management of HCV-infected individuals. Since HCV replication cannot randomly occur at lipid droplets produced in liver cells but relies instead on active core- and DGAT1-mediated lipid droplet formation, pharmaceutical inhibitors of DGAT1, currently in early clinical trials for obesity-associated diseases, may serve as novel antiviral drug candidates for HCV-infected individuals. The goal of this proposal is to test this model and develop a molecular understanding of the role of DGAT1 in the viral life cycle. We propose 1) to define the role of DGAT enzymes in HCV replication. We will establish Huh7.5 hepatoma cells lines in which expression of DGAT1 or its sister enzyme DGAT2 is stably knocked down by RNAi. These cell lines are unique tools to study the function and architecture of DGAT1- or DGAT2- generated lipid droplets during HCV infection. 2) To characterize the interaction between core and DGAT1. We observed that core physically interacts with DGAT1 and hypothesize that this interaction recruits core onto the surface of newly formed lipid droplets generated by DGAT1. We will test this hypothesis by identifying the DGAT1-interacting domain in core and examining effects of mutations in this domain on HCV assembly at lipid droplets. We will also examine whether the core/DGAT1 interaction is relevant for the localization of nonstructural protein NS5A to lipid droplets. 3) To study the mechanism of core-mediated lipid droplet accumulation. We will test whether core binding to DGAT1 activates the catalytic activity of DGAT1 to induce lipid droplet accumulation. We will also test whether core at the surface of DGAT1-generated lipid droplets prevents turnover of these droplets thereby inducing an accumulation in cells. Finally, we speculate that DGAT1-generated lipid droplets have a special interaction with mitochondria and transfer core from their surface onto the outer mitochondrial membrane, where they cause steatosis. Collectively, our studies will bring novel and important molecular insight into the mechanisms and functions of core-induced lipid droplet accumulation during HCV infection.
We seek to identify and characterize novel regulatory mechanisms controlling Hepatitis C Virus (HCV) replication that might be exploited as new therapeutic targets. Our proposed studies characterize the role of a novel cofactor of the capsid protein core, DGAT1, in the HCV life cycle. These studies are directly relevant to HCV pathogenesis and may contribute to the development of novel antiviral drugs that will address public need.
| Ramage, Holly R; Kumar, G Renuka; Verschueren, Erik et al. (2015) A combined proteomics/genomics approach links hepatitis C virus infection with nonsense-mediated mRNA decay. Mol Cell 57:329-340 |
| Webster, Brian; Ott, Melanie; Greene, Warner C (2013) Evasion of superinfection exclusion and elimination of primary viral RNA by an adapted strain of hepatitis C virus. J Virol 87:13354-69 |
| Herker, Eva; Ott, Melanie (2012) Emerging role of lipid droplets in host/pathogen interactions. J Biol Chem 287:2280-7 |
| Harris, Charles; Herker, Eva; Farese Jr, Robert V et al. (2011) Hepatitis C virus core protein decreases lipid droplet turnover: a mechanism for core-induced steatosis. J Biol Chem 286:42615-25 |
| Herker, Eva; Ott, Melanie (2011) Unique ties between hepatitis C virus replication and intracellular lipids. Trends Endocrinol Metab 22:241-8 |
