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

脂质膜在RNA病毒复制中的重要性已被广泛认识，但我们对其在这一过程中的精确化学组成和功能的了解仍然很少。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化和生物物理特性。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）改变去氨甾醇（胆固醇生物合成的倒数第二种中间体）的丰度，并使其定位于发生RNA复制的特殊膜中。由于减少稳态RNA复制，从细胞中消耗去氨甾醇具有显著的抗病毒作用。外源性去氨甾醇对这种作用的拯救不能完全由胆固醇来概括。基于这些发现，我们提出了一个模型，在这个模型中，位于复制膜上的去氨甾醇对RNA的高效复制很重要，而HCV干扰胆固醇的生物合成途径，促进去氨甾醇在该区域的积累。在这里，我们提出的目标旨在阐明HCV改变去氨甾醇稳态的独特机制，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，我们将探讨丙型肝炎病毒影响去氨甾醇稳态的机制，重点关注丙型肝炎病毒对DHCR24的影响，DHCR24是将去氨甾醇转化为胆固醇的酶。我们最近发现一种翻译后修饰的DHCR24出现在hcv感染的细胞中，并且活性的NS3-4A蛋白酶足以产生这种物种。我们在蛋白质的N端附近绘制了可能的裂解位点。因此，在Aim 1中，我们将研究这种切割事件是否会影响DHCR24的酶活性、稳定性或定位。在目标2中，我们将开发蛋白质脂质体和支持脂质双分子层系统作为模型，我们可以研究膜内的活性HCV复制酶，我们可以控制和研究膜内的脂质含量。由于去氨甾醇与胆固醇的区别仅在于碳24上存在烯烃，因此它对丙型肝炎病毒复制的独特影响是一个有趣的例子，即使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。HCV调节膜中脂质含量的机制构成了一类新的病毒-宿主相互作用。

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.