Characterization of flavovirus NS4A induced autophagy

黄病毒 NS4A 诱导自噬的表征

基本信息

批准号：
9541037
负责人：
ZAHRA ZAKERI
金额：
$ 0.79万
依托单位：
QUEENS COLLEGE
依托单位国家：
美国
项目类别：
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/9541037
关键词：
ATF6 gene 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 Cytoprotection Data Dengue Dengue Virus Disease Down-Regulation Epithelial Cells FRAP1 gene 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 XBP1 gene Yeasts biological adaptation to stress cancer cell experimental study insight member mortality mutant response sensor therapeutic target

项目摘要

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.

描述（由申请人提供）：肝细胞和肾小管上皮细胞倾向于在解决急性相后很长时间会脱落黄病毒。在这些细胞中，黄病毒在自噬小体中复制，该病毒被病毒上调。该机制未知。我们发现登革热-2或Modoc（鼠Flavivivirus）NS4A是上调自噬的唯一病毒蛋白，并且自噬对上皮细胞中的病毒复制很重要。 NS4A诱导的自噬可保护被感染的细胞免受死亡，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，病毒产生的增加以及持续感染的建立。我们假设NS4A插入ER会诱导ER应力，从而导致ROS诱导。然后，ROS增加了ATM途径，从而下调M-TOR并导致自噬。我们对线性途径的假设肯定过于简化，但是从一个简单的模型开始，并扩展到并发症将比尝试潜在的压倒性分析更有生产力。对于这些研究，我们建议：目标I：表征导致病毒感染后自噬的细胞和病毒成分。我们将首先询问我们看到的登革热2和NS4A是否在这种登革热菌株中是独一无二的，还是NS4A在所有形式的登革热中都相似地发挥作用。答：确定对所有登革热菌株的自动噬和随后保护细胞免受死亡的影响？ B：确定负责诱导自噬的登革热NS4A的域。通过发现该分子的哪一部分负责这种诱导，我们可以尝试阻止它，从而干扰病毒生存和持久的能力。 AIM II：研究登革热诱导自噬的机制。在这里，我们将测试假设的每个步骤。我们将：答：确认ATM和M-TOR途径在NS4A诱导的自噬中的参与。 B：确定NS4A表达是否导致ER应力。 C：确定NS4A感染是否导致ROS产生。 D：确定负责ER应力的NS4A的域。 E：使用酵母作为互补模型来确定NS4A介导的自噬所涉及的调节途径。我们的发现应导致对黄病毒在其宿主中如何持续的理解，从而提出减少病毒载荷和持久性的目标和潜在手段。此外，由于我们直接研究了一种调节自噬的手段，因此这些研究应导致对激活和抑制自噬的手段的新见解，这是细胞生物学的重要问题，并且在许多其他疾病中，自动噬是至关重要的其他疾病中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。