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: 10078255
• 负责人: Priscilla Li-ning Yang
• 金额: --
• 依托单位: HARVARD MEDICAL SCHOOL
• 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 2021-06-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10078255
• 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已得到广泛认可，但我们对其精确化学成分和在此过程中的功能的了解仍然知之甚少，尽管许多研究已经能够记录病毒诱导的宿主酶表达的变化。尽管RNA负责脂质生物合成和代谢，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，病毒如何组装这种特殊的膜，特定的脂质如何决定RNA的生化和生物物理特性。这些膜，以及我们发现丙型肝炎病毒 (HCV) 会改变胆固醇生物合成中倒数第二个中间体的丰度，并导致其定位于发生 RNA 复制的特殊膜中。由于稳态RNA复制减少，细胞具有显着的抗病毒作用。胆固醇不能完全重现外源性去氨甾醇的这种作用。其中位于复制膜中的去莫甾醇对于有效的 RNA 复制非常重要，并且 HCV 扰乱胆固醇生物合成途径以促进去莫甾醇在该区域的积累。在此，我们提出旨在阐明 HCV 改变去莫甾醇稳态的独特机制的目标。并建立实验系统，使我们能够在化学定义的条件下探究特定脂质对 HCV RNA 复制的影响。在目标 1 中，我们将探讨其机制。 HCV 影响去莫甾醇稳态，重点关注 HCV 对 DHCR24 的影响，DHCR24 是将去莫甾醇转化为胆固醇的酶，我们最近发现 DHCR24 的翻译后修饰形式出现在 HCV 感染的细胞中，并且活性 NS3-4A 蛋白酶就足够了。为了生成这个物种，我们已经绘制了蛋白质 N 末端附近的可能切割位点，因此，在目标 1 中，我们。将检查这种裂解事件是否影响 DHCR24 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，我们可以了解其脂质含量。由于去氨甾醇与胆固醇的区别仅在于碳 24 处存在烯烃，因此它对 HCV 复制的独特影响是一个有趣的例子，即使脂质结构看似细微的变化也可能产生影响。对膜相关生物过程的广泛影响 HCV 调节发生复制的膜中脂质含量的机制构成了一类新型的病毒-宿主相互作用。