Characterization of flavovirus NS4A induced autophagy

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一段时间内倾向于脱落黄病毒。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。我们发现登革热-2或Modoc（一种鼠黄病毒）NS4A是唯一上调自噬的病毒蛋白，并且自噬对病毒在上皮细胞中的复制很重要。ns4a诱导的自噬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。我们拟探讨NS4A如何诱导自噬、增加病毒产生和建立持续感染。我们假设NS4A插入内质网诱导内质网应激，从而诱导ROS。这种增加的ROS随后激活ATM通路，从而下调M-TOR并导致自噬。我们对线性路径的假设当然过于简化了，但从一个简单的模型开始，扩展到复杂的模型，将比尝试一个潜在的压倒性的分析更有成效。对于这些研究，我们提出：目的1：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型和NS4A中看到的是这种登革热菌株所特有的，还是NS4A在所有形式的登革热中都有类似的功能。答：确定登革热-2产生的自噬和随后的细胞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。通过找出这个分子的哪一部分负责这种诱导，我们就可以尝试阻止它，从而干扰病毒生存和持续的能力。目的二：探讨登革热诱导自噬的机制。在这里，我们将检验假设的每一步。我们将：A：确认ATM和M-TOR通路参与ns4a诱导的自噬。B：确定NS4A表达是否导致内质网应激。C：判断NS4A感染是否导致ROS产生。D：确定负责内质网应激的NS4A结构域。E：以酵母为补充模型，确定ns4a介导的自噬参与的调控途径。我们的发现将有助于了解黄病毒如何在宿主体内持续存在，从而提出降低病毒载量和持久性的靶标和潜在方法。此外，由于我们直接研究了自噬调节的手段，这些研究应该会导致对激活和抑制自噬手段的新见解，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。

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.