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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（由申请方提供）：肝细胞和肾小管上皮细胞倾向于在急性期消退后很长时间内脱落黄病毒。在这些细胞中，黄病毒在自噬体中复制，自噬体被病毒上调。其机制尚不清楚。我们发现，登革-2或莫多克（鼠黄病毒）NS 4A是唯一的病毒蛋白上调自噬，自噬是重要的病毒复制上皮细胞。NS 4A诱导的自噬保护感染细胞免于死亡，这是建立病毒持久性的先决条件。我们建议探索NS 4A如何诱导自噬，增加病毒产量，并建立持续感染。我们假设NS 4A插入ER诱导ER应激，导致ROS的诱导。这增加的ROS然后激活ATM通路，从而下调M-TOR并导致自噬。我们的线性路径假设当然过于简化，但从一个简单的模型开始，扩展到包括复杂性，将比尝试潜在的压倒性分析更有成效。对于这些调查，我们建议：目的一：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2型和NS 4A中看到的是这种登革病毒株所特有的，还是NS 4A在所有形式的登革病毒中的功能都是相似的。答：确定自噬和随后的保护细胞免于死亡产生的登革热2是普遍适用于所有登革热菌株？B：鉴定负责诱导自噬的登革NS 4A的结构域。通过找到该分子的哪个部分负责这种诱导，我们可以尝试阻止它，从而干扰病毒的生存和持续能力。目的II：阐明登革诱导自噬的机制。在这里，我们将测试我们假设的每一步。我们将：A：确认ATM和M-TOR通路参与NS 4A诱导的自噬。B：确定NS 4 A表达是否导致ER应激。C：确定NS 4A感染是否导致ROS产生。D：鉴定负责ER应激的NS 4A的结构域。E：使用酵母作为补充模型来确定参与NS 4A介导的自噬的调控途径。我们的研究结果应该导致了解黄病毒如何在其宿主中持续存在，从而提出了降低病毒载量和持久性的目标和潜在手段。此外，由于我们直接研究了调节自噬的方法，这些研究应该会对激活和抑制自噬的方法产生新的见解，这是细胞生物学中的一个重要问题，也是许多其他疾病的潜在治疗靶点，其中自噬对细胞存活至关重要，例如神经退行性疾病和癌症。