Non-vesicular lipid transport by poxvirus A6 protein

真核细胞由独立的膜结合细胞器组成，具有不同的脂质成分。脂质成分通过囊泡运输以及鲜为人知的脂质转移蛋白（LTP）介导的非囊泡运输来维持。重要的是要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、病毒粒子产生和退出的策略。发现这些策略不仅可以揭示抗病毒药物开发的关键病毒复制步骤，还可以提供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层脂质双分子层，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。新月形膜的起源和生物发生使病毒学家困惑了半个多世纪，尽管最近的研究表明，新月形膜可能以一种独立于内质网的囊泡运输的方式来源于内质网（ER）。五种病毒蛋白，统称为病毒膜组装蛋白（VMAPs），已被发现是必不可少的新月膜的生物发生。牛痘病毒的A6蛋白是VMAPs的关键成员，是我们发现并深入研究的。在最近的研究中，我们通过求解A6蛋白的N-和C-结构域的晶体结构，实现了A6蛋白结构分析的突破。更重要的是，我们的结构和生化研究表明，C结构域是一种新型的脂质结合蛋白，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。这使我们提出了一个创新的假设，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介导的非囊泡性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检验我们的新假设。目的1。测定a6aim 2结合脂质的特异性和化学计量学。目的：探讨A6脂质结合在病毒膜生物发生中的作用。确定A6 n结构域调控c结构域脂质结合的结构基础。

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.