Elucidating the role of lipids in positive-sense RNA virus infection.

细胞内病原体严重依赖宿主细胞代谢途径进行复制和/或持续感染。我们假设正义RNA病毒需要共同的脂质代谢途径来进行复制。丙型肝炎病毒（HCV）的复制依赖于胆固醇酯和甘油三酯的生物合成以及重新合成胆固醇和脂肪酸。HCV RNA通过内质网衍生的膜结构内的RNA复制复合物进行复制，这种膜网具有洗涤剂抗性，因为存在与脂质微域相关的脂质，即胆固醇和鞘脂。重要的是，抑制胆固醇和鞘磷脂的生物合成可以抑制细胞中的病毒RNA复制，而降低胆固醇的药物如他汀类药物可能有助于治疗成功。此外，脂肪酸合成酶（FASN）酶活性是在黄病毒科成员(包括HCV、登革热病毒（DENV）、黄热病病毒（YFV）、Usutu病毒（USUV）和西尼罗河病毒（WNV）的复制周期中产生改变膜结构所必需的。因此，抑制FASN导致病毒复制的剧烈抑制。然而，感染黄病毒科不同成员的哺乳动物细胞脂质代谢的确切变化目前尚不清楚。我们最近深入研究了hcv诱导的脂质组成的变化，并对全细胞提取物和亚细胞区室进行了定量的散弹枪脂质组学研究。我们的研究结果表明，HCV感染降低了中性脂与膜脂的比例。此外，hcv感染的细胞具有较高的磷脂酰胆碱和甘油三酯的相对丰度，具有较长的脂肪酸酰基链，并且C18脂肪酸（最显著的是油酸）的利用率显著增加。因此，脂肪酸延长酶和去饱和酶的耗尽会损害HCV的复制和/或颗粒的组装和释放。综上所述，我们的初步结果表明，HCV诱导的宿主细胞脂质代谢的复杂重塑可以增强病毒复制和后代的产生。在这里，我们建议进行hcv相关病毒（如DENV， YFV和Zika病毒（ZIKV））的脂质组学研究，以比较和对比黄病毒科病毒的代谢重布线。这些研究将揭示脂质重塑的异同，并探讨它们与病毒复制的相关性。其次，我们将详细研究特定脂肪酸延长酶和去饱和酶及其酶产物在病毒RNA复制囊泡形成中的作用。最终目的是阐明多不饱和脂肪酸在病毒粒子形态发生中的作用。

Intracellular pathogens critically depend on host cell metabolic pathways for replication and/or persistent infection. We hypothesize that positive-sense RNA viruses require common lipid metabolic pathways for their replication. Hepatitis C virus (HCV) replication depends on cholesterol ester and triglyceride biosynthesis as well as de novo cholesterol and fatty acid synthesis. The HCV RNA is replicated by RNA replication complexes within ER-derived membrane structures termed the membranous web that is detergent-resistant due to the presence of lipids that are associated with lipid microdomains, namely cholesterol and sphingolipids. Importantly, inhibition of cholesterol and sphingomyelin biosynthesis suppresses viral RNA replication in cells and cholesterol-lowering agents such as statins may be beneficial for therapeutic success. In addition, fatty acid synthase (FASN) enzymatic activity is required for the creation of altered membrane structures in the replication cycle of Flaviviridae family members including HCV, dengue virus (DENV), yellow fever virus (YFV), Usutu virus (USUV), and West Nile virus (WNV). Accordingly, inhibition of FASN leads to a drastic inhibition of viral replication. However, the precise changes in the lipid metabolism of mammalian cells infected with different members of the Flaviviridae family are currently unknown. We recently investigated in-depth the HCV-induced changes in the lipid composition and performed quantitative shotgun lipidomic studies of whole cell extracts and subcellular compartments. Our results indicate that HCV infection reduces the ratio of neutral to membrane lipids. In addition, HCV-infected cells had a higher relative abundance of phosphatidylcholines and triglycerides with longer fatty acyl chains and a striking increased utilization of C18 fatty acids, most prominently oleic acid. Accordingly, depletion of fatty acid elongases and desaturases impaired HCV replication and/or assembly and release of particles. Taken together our preliminary results demonstrate the complex remodeling of the host cell lipid metabolism induced by HCV to enhance both virus replication and progeny production.Here, we propose to perform lipidomic studies of HCV-related viruses such as DENV, YFV, and Zika virus (ZIKV) to compare and contrast the metabolic rewiring by viruses of the Flaviviridae family. These studies will uncover similarities and differences in lipid remodeling that will be probed for their relevance for viral replication. Second, we will study in great mechanistic detail the function of specific fatty acid elongases and desaturases and their enzymatic products in the formation of viral RNA replication vesicles. The final aim will elucidate the role of polyunsaturated fatty acids in virion morphogenesis.