How Hepatitis C Virus Regulates Desmosterol to Affect RNA Replication: a New Virus-Host Interaction

丙型肝炎病毒如何调节去莫甾醇影响 RNA 复制：一种新的病毒-宿主相互作用

• 批准号: 10433794
• 负责人: Priscilla Li-ning Yang
• 金额: $ 23.62万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433794
• 关键词: Affect; Alkenes; Antiviral Agents; Binding Sites; Biochemical; Biological Assay; Biological Process; Biophysics; Carbon; Cells; Chemicals; Cholesterol; Cleaved cell; Cryoelectron Microscopy; Data; Desmosterol; Disease; Engineering; Enzymes; Equine mule; Event; Experimental Models; Fractionation; Genes; Goals; Hepatitis C virus; Homeostasis; Investigation; Isotope Labeling; Knowledge; Lipid Bilayers; Lipids; Locales; Maps; Membrane; Membrane Fluidity; Membrane Lipids; Membrane Structure and Function; Microscopy; Modeling; Modification; Mutation; N-terminal; Nonstructural Protein; Pathway interactions; Peptide Hydrolases; Physiological; Post-Translational Protein Processing; Process; Production; Property; Proteins; RNA; RNA Viruses; RNA replication; Signal Transduction; Site; System; Testing; Translating; Viral; Virus; Virus Replication; Work; base; biophysical properties; cholesterol biosynthesis; design; enzyme activity; experimental study; fluidity; lipid biosynthesis; lipid metabolism; lipid structure; membrane model; novel; pathogenic virus; prevent; proteoliposomes; replicase; viral RNA; virus host interaction

The importance of lipid membranes in the replication of RNA viruses is widely appreciated, yet our understanding of their precise chemical composition and function in this process remains poorly understood. While many studies have been able to document virus-induced changes in the expression of host enzymes responsible for lipid biosynthesis and metabolism, few studies have been able to translate these findings into chemical knowledge of the lipid species present in the membranes where RNA replication occurs, how the virus assembles this specialized membrane, how particular lipids dictate the biochemical and biophysical properties of these membranes, and how these properties in turn affect viral RNA replication. We discovered that hepatitis C virus (HCV) alters the abundance of desmosterol, a penultimate intermediate in cholesterol biosynthesis, and causes it to localize in the specialized membranes where RNA replication occurs. Depletion of desmosterol from the cell has a significant antiviral effect due to decreased steady-state RNA replication. Rescue of this effect by exogenous desmosterol cannot be fully recapitulated by cholesterol. Based on these findings, we have proposed a model in which desmosterol localized in the replication membrane is important for efficient RNA replication, and HCV perturbs the cholesterol biosynthetic pathway to promote accumulation of desmosterol at this locale. Here, we propose Aims designed to elucidate the unique mechanism(s) whereby HCV alters desmosterol homeostasis and to establish experimental systems that allow us to interrogate the effects of specific lipids on HCV RNA replication under chemically defined conditions. In Aim 1, we will probe the mechanisms by which HCV affects desmosterol homeostasis, focusing on HCV’s effects on DHCR24, the enzyme that converts desmosterol to cholesterol. We recently found that a post-translationally modified form of DHCR24 appears in HCV-infected cells and that the active NS3-4A protease is sufficient to generate this species. We have mapped the likely cleavage site near the N- terminus of the protein. Therefore, in Aim 1, we will examine whether this cleavage event affects the enzymatic activity, stability, or localization of DHCR24. In Aim 2, we will develop proteoliposome and supported lipid bilayer systems as models in which we can study the active HCV replicase within a membrane whose lipid content we can control and study. Since desmosterol differs from cholesterol only by the presence of an alkene at carbon 24, its distinct effects on HCV replication are an intriguing example that even seemingly subtle changes in lipid structure may have profound effects on membrane-associated biological processes. HCV’s mechanism for tuning lipid content in the membrane where replication occurs constitutes a novel class of virus-host interactions.

脂质膜在RNA病毒复制中的重要性得到了广泛的认可，但我们对它们在这一过程中的精确化学组成和功能的理解仍然知之甚少。虽然许多研究已经能够记录病毒诱导的负责脂质生物合成和代谢的宿主酶表达的变化，但很少有研究能够将这些发现转化为RNA复制发生的膜中存在的脂质种类的化学知识，病毒如何组装这种专门的膜，特定的脂质如何决定这些膜的生物化学和生物物理性质，以及这些特性如何反过来影响病毒RNA复制。我们发现，丙型肝炎病毒（HCV）改变了桥甾醇的丰度，胆固醇生物合成的倒数第二个中间体，并导致其定位在RNA复制发生的专门膜。由于稳态RNA复制减少，细胞中链甾醇的消耗具有显著的抗病毒作用。外源性链甾醇对这种作用的拯救不能完全被胆固醇重现。基于这些发现，我们提出了一个模型，其中桥甾醇位于复制膜是重要的有效的RNA复制，和HCV干扰胆固醇生物合成途径，以促进积累桥甾醇在这个位置。在这里，我们提出的目的，旨在阐明独特的机制（S），从而HCV改变桥甾醇稳态，并建立实验系统，使我们能够询问特定的脂质对HCV RNA复制的化学条件下的影响。在目标1中，我们将探讨HCV影响桥甾醇稳态的机制，重点是HCV对DHCR 24（将桥甾醇转化为胆固醇的酶）的影响。我们最近发现，HCV感染的细胞中出现了DHCR 24的后修饰形式，并且活性NS 3 -4A蛋白酶足以产生这种物质。我们已经定位了蛋白质N端附近可能的切割位点。因此，在目的1中，我们将检查这种切割事件是否影响DHCR 24的酶活性、稳定性或定位。在目标2中，我们将开发蛋白脂质体和支持的脂质双层系统作为模型，在该模型中，我们可以研究膜内的活性HCV复制酶，其脂质含量可以控制和研究。由于链甾醇与胆固醇的区别仅在于碳24位存在烯烃，因此其对HCV复制的独特影响是一个有趣的例子，即使是脂质结构中看似细微的变化也可能对膜相关的生物过程产生深远的影响。丙型肝炎病毒的机制，调整脂质含量的膜复制发生构成了一类新的病毒-宿主相互作用。