Non-vesicular lipid transport by poxvirus A6 protein

痘病毒 A6 蛋白的非囊泡脂质转运

• 批准号: 9379762
• 负责人: Junpeng Deng
• 金额: $ 23.44万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-05 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9379762
• 关键词: Address; Antiviral Agents; Architecture; Binding; Binding Proteins; Biochemical; Biogenesis; Biological Assay; Cardiovascular Diseases; Cell Line; Cell physiology; Cells; Cellular Membrane; Complement; Crystallization; Dangerousness; Data; Development; Diabetes Mellitus; Endoplasmic Reticulum; Eukaryotic Cell; Fluorescence; Genome; Glycerol; Goals; Human Pathology; Hydrophobicity; In Vitro; Individual; Knowledge; Length; Lipid Bilayers; Lipid Binding; Lipids; Malignant Neoplasms; Mediating; Membrane; Molecular Conformation; Monkeypox virus; Mutation; N Domain; Neurodegenerative Disorders; Obesity; Organelles; Pathogenicity; Phospholipids; Play; Population; Poxviridae; Process; Production; Proteins; Recombinants; Roentgen Rays; Role; Site-Directed Mutagenesis; Smallpox Viruses; Specificity; Structure; Testing; Transport Process; Transport Vesicles; Vaccinia virus; Vesicle Transport Pathway; Viral; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; base; innovation; insight; lipid transfer protein; lipid transport; member; membrane assembly; membrane biogenesis; monomer; mutant; novel; obligate intracellular parasite; stoichiometry; tandem mass spectrometry; virus envelope

Eukaryotic cells are organized with separate membrane-bound organelles with distinct lipid compositions. The lipid compositions are maintained through vesicular transport as well as the less-understood, lipid-transfer protein (LTP)-mediated, nonvesicular transport. It is important to gain a better understanding of lipid transfer process, as perturbations of lipid trafficking contribute to human pathologies including cancer, neurodegenerative disorders, cardiovascular diseases, obesity and diabetes. Viruses, as obligate intracellular parasites, have evolved strategies to manipulate the cellular membranes for entry, genome replication, virion production, and exit. Uncovering these strategies will not only reveal key viral replication steps for antivirals development but also provide mechanistic insights on fundamental cellular processes. Enveloped viruses typically acquire their outer lipid bilayer by budding from cellular membranes, a process that is similar to the formation of cellular transport vesicles. Poxviruses, however, are unusual in that their primary envelope is not acquired by budding but through extending of open-ended crescent membranes. The origin and biogenesis of the crescent membranes have puzzled virologists for over half a century, albeit recent studies suggest that the crescents may derive from the endoplasmic reticulum (ER) in a manner that is independent of vesicular transport from the ER. Five viral proteins, collectively termed viral membrane assembly proteins (VMAPs), have been found to be essential for the biogenesis of crescent membranes. The A6 protein of vaccinia virus is a key member of the VMAPs, which we discovered and have studied intensively. In recent studies, we achieved a breakthrough in structural analysis of the A6 protein by solving the crystal structures of both its N- and C- domains. Even more importantly, our structural and biochemical studies indicate that the C- domain is a novel lipid binding protein with an unusually high binding capacity for glycerol-phospholipids and that the N-domain regulates lipid binding. These led us to the innovative hypotheses that A6 is a lipid-transfer protein (LTP) and that poxviruses obtain their primary envelope by mimicking or hijacking the cellular LTP-mediated nonvesicular lipid transport process. We propose the following exploratory studies to test our novel hypotheses. Aim 1. To determine the specificity and stoichiometry of the lipids bound by A6 Aim 2. To determine the role of lipid binding of A6 in viral membrane biogenesis Aim 3. To determine the structural basis by which A6 N-domain regulates C-domain for lipid binding.

真核细胞由具有不同脂质成分的独立膜结合细胞器组成。脂质成分通过囊泡运输以及不太了解的脂质转移蛋白（LTP）介导的非囊泡运输来维持。更好地了解脂质转移过程非常重要，因为脂质运输的扰动会导致人类病理，包括癌症、神经退行性疾病、心血管疾病、肥胖和糖尿病。病毒作为专性细胞内寄生虫，已经进化出操纵细胞膜进入、基因组复制、病毒体产生和退出的策略。揭示这些策略不仅将揭示抗病毒药物开发的关键病毒复制步骤，还将提供对基本细胞过程的机制见解。有包膜病毒通常通过从细胞膜出芽来获得其外部脂质双层，这一过程类似于细胞运输囊泡的形成。然而，痘病毒的不同寻常之处在于，它们的初级包膜不是通过出芽获得的，而是通过开放式新月膜的延伸获得的。半个多世纪以来，新月膜的起源和生物发生一直困扰着病毒学家，尽管最近的研究表明，新月膜可能以一种独立于内质网囊泡运输的方式衍生自内质网（ER）。已发现五种病毒蛋白，统称为病毒膜组装蛋白（VMAP），对于新月膜的生物发生至关重要。痘苗病毒的A6蛋白是VMAPs的关键成员，我们发现并深入研究了它。在最近的研究中，我们通过解析 A6 蛋白 N 和 C 结构域的晶体结构，在结构分析方面取得了突破。更重要的是，我们的结构和生化研究表明，C 结构域是一种新型脂质结合蛋白，对甘油磷脂具有异常高的结合能力，而 N 结构域调节脂质结合。这些使我们提出了创新假设，即 A6 是一种脂质转移蛋白 (LTP)，痘病毒通过模仿或劫持细胞 LTP 介导的非囊泡脂质转运过程来获得其初级包膜。我们提出以下探索性研究来检验我们的新假设。目标 1. 确定 A6 结合的脂质的特异性和化学计量学 目标 2. 确定 A6 的脂质结合在病毒膜生物合成中的作用 目标 3. 确定 A6 N 结构域调节 C 结构域脂质结合的结构基础。