Regulation of reovirus induced apoptosis

描述(申请人提供)：在这项提案中，我们试图了解呼肠孤病毒诱导细胞凋亡的潜在机制，以及如何调节这一过程以优化培养细胞和体内的病毒复制。细胞凋亡是一种进化上保守的机制，通过这种机制，细胞在发育过程中被移除，正常组织的动态平衡得以维持。在病理条件下，抑制细胞凋亡可导致癌症，而自身免疫或诱导细胞凋亡可导致组织损伤。许多致病的人类病毒，如登革热病毒、流感病毒和西尼罗河病毒，通过诱导细胞凋亡而导致疾病。一些病毒在感染周期的后期诱导细胞凋亡，这使得病毒能够在宿主内传播。关于病毒如何调控细胞凋亡诱导的起始和时间，人们知之甚少。哺乳动物呼肠孤病毒是鉴定病毒致病一般机制的重要模型。呼肠孤病毒通过诱导细胞凋亡造成病理损害(心肌炎和脑炎)。此外，这些病毒优先针对转化的细胞系，由于它们对人类的安全性和对多种肿瘤类型的活性(它们目前正在几个II/III期临床试验中进行测试)，作为溶瘤药物的前景越来越大。重要的是，病毒诱导的细胞凋亡是肿瘤细胞杀伤的主要机制。在这里，我们将使用呼肠孤病毒模型来剖析病毒诱导细胞凋亡的过程及其调控，重点关注病毒结构蛋白？1，它在病毒进入细胞时起着渗透细胞膜的作用，并在感染周期的后期诱导细胞凋亡。提出了两个具体目标。首先，我们将确定外衣壳蛋白？1诱导细胞凋亡的机制。这些实验将检验这一假设，即？1的C末端片段是一种病毒孢菌素，可以直接穿透细胞膜，包括质膜，允许分子泄漏到胞浆中，激活细胞应激途径，诱导细胞凋亡。在第二部分中，将定义调节细胞凋亡、细胞毒性和致病机制的？1的翻译后修饰。这些实验将验证这一假设，即感染细胞胞浆中的蛋白水解性切割和泛素化的Free？1调节病毒的释放、细胞毒性和致病性。总而言之，这些研究将加强对病毒诱导的细胞杀伤的基本了解，并可能促进用于溶瘤应用的改进的呼肠孤病毒载体的开发，并导致针对凋亡途径的新的抗病毒疗法。

DESCRIPTION (provided by applicant): In this proposal we seek to understand the underlying mechanisms by which reoviruses induce apoptosis in cells and how this process is regulated to optimize viral replication in cultured cells and in vivo. Apoptosis is an evolutionarily conserved mechanism by which cells are removed during development and normal tissue homeostasis is maintained. Under pathologic conditions, suppression of apoptosis can lead to cancer and autoimmunity or conversely induction of apoptosis can lead to tissue injury. Many pathogenic human viruses, e.g., dengue virus, influenza virus, and West Nile virus, cause disease by inducing apoptosis. Some viruses induce apoptosis late in the infectious cycle, which allows the virus to spread within the host. Little is known about how viruses regulate the initiation and timing of apoptosis induction. Mammalian reoviruses are important models for identification of general pathogenic mechanisms by which viruses cause disease. Reoviruses cause pathologic damage (myocarditis and encephalitis) by inducing apoptosis. In addition, these viruses preferentially target transformed cell lines and are showing increasing promise as oncolytic agents because of their safety in humans and activity against a broad range of tumor types (they are currently being tested in several phase II/III clinical trials). Importantly, the primary mechanism of tumor cell killing is by virus-induced apoptosis. Here we will use the reovirus model to dissect the process of virus-induced apoptosis and its regulation by focusing on the viral structural protein, ?1, which functions to permeabilize cell membranes during virus entry and induces apoptosis later in the infectious cycle. Two specific aims are proposed. In the first, mechanisms by which outer-capsid protein ?1 induces apoptosis will be determined. These experiments will test the hypothesis that a C-terminal fragment of ?1 is a viroporin that can directly permeabilize cellular membranes, including the plasma membrane, allowing leakage of molecules into the cytosol that activate cellular stress pathways, inducing apoptosis. In the second, post-translational modifications of ?1 that regulate the induction of apoptosis, cytotoxicity, and pathogenesis will be defined. These experiments will test the hypothesis that proteolytic cleavage and ubiquitination of free ?1 in the cytosol of infected cells regulates virus release, cytotoxicity, and pathogenicity. Collectively, these studies will enhance a basic understanding of virus-induced cell killing and may foster development of improved reovirus vectors for oncolytic applications and lead to new antiviral therapeutics that target apoptotic pathways.