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识，但是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很少了解。 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这些特性又如何影响病毒RNA复制。我们发现丙型肝炎病毒（HCV）改变了胆固醇生物合成中倒数第二个中间体的抽象，并导致其定位在发生RNA复制的专用膜中。由于稳态RNA的复制减少，从细胞中耗尽了Desmasterol的抗病毒作用显着。胆固醇不能完全概括外源性去甲醇来挽救这种作用。基于这些发现，我们提出了一个模型，在该模型中，在复制膜中定位的desmasterol对于有效的RNA复制很重要，而HCV伴随着胆固醇生物合成途径，以促进Desmasterol在该网站上的积累。在这里，我们提出的目的旨在阐明HCV改变desmasterol稳态的独特机制，并建立实验系统，使我们能够在化学定义的条件下询问特定脂质对HCV RNA复制的影响。在AIM 1中，我们将探测HCV影响desmasterol稳态的机制，重点是HCV对DHCR24的影响，DHCR24是将desmasterol转化为胆固醇的酶。我们最近发现，在HCV感染的细胞中出现了跨化后修饰的DHCR24形式，并且活性NS3-4A蛋白酶足以生成该物种。我们已经绘制了蛋白质N末端附近的可能性切割位点。因此，在AIM 1中，我们将检查该裂解事件是否影响DHCR24的酶活性，稳定性或定位。在AIM 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，我们可以在该模型中研究我们可以控制和研究的脂质含量的膜中的活性HCV复制酶。由于仅通过碳24处的烯烃存在与胆固醇的不同之处，因此其对HCV复制的独特作用是一个有趣的例子，即即使是脂质结构看似微妙的变化，也可能对膜相关的生物学过程产生深远的影响。 HCV在复制发生的膜上调整脂质含量的机制构成了一种新型的病毒宿主相互作用。