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组成的独立膜结合细胞器组成。脂质成分通过囊泡转运以及较少了解的脂质转运蛋白（LTP）介导的非囊泡转运来维持。重要的是更好地了解脂质转移过程，因为脂质运输的扰动有助于人类病理学，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物，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。包膜病毒通常通过从细胞膜出芽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，痘病毒是不寻常的，因为它们的初级包膜不是通过出芽获得的，而是通过开放的新月形膜的延伸获得的。新月体膜的起源和生物发生已经困扰了病毒学家超过半个世纪，尽管最近的研究表明新月体可能以独立于囊泡运输的方式来自内质网（ER）。已发现五种病毒蛋白，统称为病毒膜组装蛋白（VMAP），对新月膜的生物发生至关重要。A6蛋白是我们发现并深入研究的VMAPs的重要成员。在最近的研究中，我们通过解决其N-和C-结构域的晶体结构，在A6蛋白的结构分析方面取得了突破。甚至更重要的是，我们的结构和生物化学研究表明，C-结构域是一种新的脂质结合蛋白，具有异常高的甘油-磷脂结合能力，N-结构域调节脂质结合。这些使我们创新的假设，A6是一种脂质转运蛋白（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获得其初级包膜。我们提出以下探索性研究来验证我们的新假设。目标1.确定A6 Aim 2结合脂质的特异性和化学计量。确定A6的脂质结合在病毒膜生物合成中的作用。确定A6 N-结构域调节C-结构域与脂质结合的结构基础。