Characterization of flavovirus NS4A induced autophagy

黄病毒 NS4A 诱导自噬的表征

• 批准号: 8099376
• 负责人: ZAHRA ZAKERI
• 金额: $ 45.13万
• 依托单位: QUEENS COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2017-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8099376
• 关键词: ATM Signaling Pathway; ATM activation; Acute; Amino Acids; Antithymoglobulin; Autophagocytosis; Autophagosome; Biological Models; Cell Death; Cell Survival; Cells; Cellular biology; Cessation of life; Chemicals; Child; Culicidae; Data; Dengue; Dengue Virus; Disease; Down-Regulation; Epithelial Cells; Flavivirus; Flavivirus Infections; Genes; Helper-Inducer T-Lymphocyte; Hepatocyte; Hospitalization; Infection; Investigation; Kidney; Lead; Link; Malignant Neoplasms; Mediating; Methods; Modeling; Morbidity - disease rate; Mus; N-terminal; Neurodegenerative Disorders; Neurons; Oxidative Stress; Pathway interactions; Phase; Production; Protein Region; Proteins; RNA Splicing; Regulatory Pathway; Southeastern Asia; Stress; Suicide prevention; System; Tail; Testing; Therapeutic; Tubular formation; Up-Regulation; Viral; Viral Load result; Viral Proteins; Virus; Virus Diseases; Virus Replication; Western Blotting; Work; Yeasts; biological adaptation to stress; cancer cell; human FRAP1 protein; insight; member; mortality; mutant; research study; response; sensor; therapeutic target

DESCRIPTION (provided by applicant): Hepatocytes and renal tubular epithelial cells tend to shed flavivirus long after the acute phase has been resolved. In these cells, flavivirus replicates in autophagosomes, which are upregulated by the virus. The mechanism is unknown. We find that Dengue-2 or Modoc (a murine flavivirus) NS4A is the sole viral protein upregulating autophagy, and that autophagy is important to virus replication in epithelial cells. NS4A-induced autophagy protects infected cells against death, a prerequisite for the establishment of viral persistence. We propose to explore how NS4A induces autophagy, increase in viral production, and establishment of persistent infection. We hypothesize that NS4A insertion into the ER induces ER stress, leading to induction of ROS. This increased ROS then activates the ATM pathways, thus downregulating M-TOR and leading to autophagy. Our hypothesis of a linear pathway is certainly oversimplified but starting with a simple model and expanding to incorporate complications will be more productive than attempting a potentially overwhelming analysis. For these investigations we propose to: AIM I: CHARACTERIZE CELLULAR AND VIRAL COMPONENTS LEADING TO AUTOPHAGY AFTER VIRAL INFECTION. We will first ask if what we see with Dengue 2 and NS4A is unique to this strain of Dengue or if NS4A functions similarly in all forms of Dengue. A: Determine if the autophagy and subsequent protection of cells from death produced by Dengue-2 is generalizable to all Dengue strains? B: Identify the domain(s) of Dengue NS4A responsible for inducing autophagy. By finding which part of this molecule is responsible for this induction we can then attempt to block it, thus interfering with the ability of the virus to survive and persist. AIM II: INVESTIGATE THE MECHANISMS BY WHICH DENGUE INDUCES AUTOPHAGY. Here we will test each step of our hypothesis. We will: A: Confirm the involvement of ATM and M-TOR pathways in NS4A-induced autophagy. B: Establish if NS4A expression leads to ER stress. C: Determine whether NS4A infection leads to ROS production. D: Identify the domain of NS4A responsible for ER stress. E: Use yeast as a complementary model to determine the regulatory pathway involved in NS4A-mediated autophagy. Our findings should lead to an understanding of how flavivirus persists in its host, thus suggesting a target and potential means of reducing viral load and persistence. Furthermore, since we directly examine a means by which autophagy is regulated, these studies should lead to new insights into the means of activating and suppressing autophagy, an important question in cell biology and a potential therapeutic target in many other diseases in which autophagy is critical for cell survival, such as in neurodegenerative diseases and cancer. PUBLIC HEALTH RELEVANCE: Dengue virus, currently the world's most common mosquito-borne illness, is the leading cause of children's hospitalization in Southeast Asia and is the cause of significant morbidity and mortality. We find that flavivirus induces autophagy, preventing suicide of the host cell, leading to increased production and persistence of virus; these activities are produced by the viral protein NS4A. We wish to analyze the mechanism by which NS4A works. Understanding the mechanism should provide a means of controlling the virus and give us another option to protect nerve cells or destroy cancer cells.

描述（由申请人提供）：肝细胞和肾小管上皮细胞在急性期结束后很长时间内往往会排出黄病毒。在这些细胞中，黄病毒在自噬体中复制，并被病毒上调。其机制尚不清楚。我们发现 Dengue-2 或 Modoc（一种鼠黄病毒）NS4A 是唯一上调自噬的病毒蛋白，并且自噬对于上皮细胞中的病毒复制非常重要。 NS4A 诱导的自噬可保护受感染细胞免于死亡，这是建立病毒持久性的先决条件。我们建议探索 NS4A 如何诱导自噬、增加病毒产量以及建立持续感染。我们假设 NS4A 插入 ER 会诱导 ER 应激，从而诱导 ROS 的产生。 ROS 的增加随后会激活 ATM 通路，从而下调 M-TOR 并导致自噬。我们对线性路径的假设肯定过于简单化，但从一个简单的模型开始并扩展到合并复杂性将比尝试潜在的压倒性分析更有成效。对于这些研究，我们建议： 目标 I：表征病毒感染后导致自噬的细胞和病毒成分。我们首先要问的是，我们在登革热 2 和 NS4A 中看到的情况是否是该登革热菌株所独有的，或者 NS4A 在所有形式的登革热中是否具有相似的功能。答：确定 Dengue-2 产生的自噬和随后的细胞免于死亡保护是否适用于所有登革热菌株？ B：确定登革热 NS4A 负责诱导自噬的结构域。通过找到该分子的哪个部分负责这种诱导，我们就可以尝试阻止它，从而干扰病毒生存和持续存在的能力。目标 II：研究登革热诱导自噬的机制。在这里，我们将检验我们假设的每一步。我们将： A：确认 ATM 和 M-TOR 通路参与 NS4A 诱导的自噬。 B：确定 NS4A 表达是否会导致 ER 应激。 C：确定 NS4A 感染是否导致 ROS 产生。 D：确定 NS4A 负责 ER 应激的域。 E：使用酵母作为补充模型来确定 NS4A 介导的自噬所涉及的调控途径。我们的研究结果应该有助于了解黄病毒如何在宿主体内持续存在，从而提出减少病毒载量和持续存在的目标和潜在方法。此外，由于我们直接检查自噬的调节方式，这些研究应该会对激活和抑制自噬的方式产生新的见解，这是细胞生物学中的一个重要问题，也是许多其他自噬至关重要的疾病的潜在治疗靶点用于细胞存活，例如神经退行性疾病和癌症。 公共卫生相关性：登革热病毒是目前世界上最常见的蚊媒疾病，是东南亚儿童住院的主要原因，也是造成严重发病和死亡的原因。我们发现黄病毒会诱导自噬，防止宿主细胞自杀，从而增加病毒的产量和持久性；这些活性是由病毒蛋白 NS4A 产生的。我们希望分析NS4A的工作机制。了解这一机制应该提供一种控制病毒的方法，并为我们提供另一种保护神经细胞或破坏癌细胞的选择。