Regulated Intramembrane Proteolysis of CREB3L1 in Innate Antiviral Response

描述（由申请人提供）：丙型肝炎病毒（HCV）感染世界人口的3%，占大多数慢性肝病病例。在美国，丙型肝炎病毒感染是导致肝功能衰竭的主要原因。由于HCV复制过程中的高突变率使得耐药病毒株快速出现，因此很难开发针对HCV蛋白的药物。目前丙型肝炎病毒感染的治疗是基于干扰素，它介导对RNA病毒感染的先天免疫反应。然而，这种治疗只是部分有效。因此，了解其他先天抗病毒反应可能揭示治疗HCV感染急需的新策略。我们最近发现了一种新的先天抗病毒反应，它在限制人类肝癌细胞的HCV感染中起重要作用。该通路由cAMP反应元件结合蛋白3-like 1 （CREB3L1）介导，其功能此前未知。CREB3L1属于一个转录因子家族，它是作为插入内质网（ER）的膜结合前体合成的，并被称为调节膜内蛋白水解（RIP）的过程激活。基于我们目前对RIP的理解，我们提出HCV在内质网中的复制导致Site-1蛋白酶（S1P）和Site-2蛋白酶（S2P）切割CREB3L1。蛋白水解裂解使CREB3L1的nh2末端片段从膜上释放出来，并进入细胞核，激活参与抗病毒反应的靶基因。这些假设将通过提案中提出的三个具体目标来检验。特异性Aim 1将确定HCV复制刺激CREB3L1切割的机制。我们将研究由HCV编码膜蛋白表达诱导的内质网应激是否会触发CREB3L1的裂解。特异性Aim 2将确定CREB3L1的S1P和s2p催化的裂解是否需要其抗病毒功能。主要方法是制造不能被这些蛋白酶切割的CREB3L1突变体，并检查突变对其抗病毒功能的影响。特异性Aim 3将通过微阵列分析鉴定抑制HCV复制的CREB3L1靶基因。如果这些具体目标得以实现，我们将贡献新的信息，这将大大提高我们对先天免疫反应的理解。这一新发现可能揭示治疗HCV感染的新药物靶点。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infects 3% of the world population and accounts for most cases of chronic liver disease. In the United States, HCV infection is the leading cause of liver failure. Drugs targeting HCV proteins are difficult to be developed owing to the high rate of mutation during HCV replication that allows quick appearance of the drug-resistant viral strains. The current treatment for HCV infection is based on interferon, which mediates an innate immune response against infection of RNA virus. However, this treatment is only partially effective. Thus, understanding other innate antiviral responses may reveal much needed new strategies to treat HCV infection. We have recently identified a novel innate antiviral response that plays an important role in limiting HCV infection in a line of human hepatoma cells. This pathway is mediated by cAMP response element binding protein 3-like 1 (CREB3L1), the function of which was previously unknown. CREB3L1 belongs to a family of transcription factors that are synthesized as membrane-bound precursors inserted in the endoplasmic reticulum (ER), and activated by a process termed regulated intramembrane proteolysis (RIP). Based on our current understanding of RIP, we propose that replication of HCV in the ER results in cleavage of CREB3L1 by Site-1 protease (S1P) and Site-2 protease (S2P). The proteolytic cleavage allows the NH2-terminal fragment of CREB3L1 to be released from the membrane and travel to the nucleus to activate its target genes involved in antiviral responses. These hypotheses will be tested by three specific aims raised in the proposal. Specific Aim 1 will determine the mechanism by which HCV replication stimulates the cleavage of CREB3L1. We will examine whether ER stress induced by expression of HCV- encoded membrane proteins triggers the cleavage of CREB3L1. Specific Aim 2 will determine whether S1P and S2P-catalyzed cleavages of CREB3L1 is required for its antiviral function. The primary approach is to make CREB3L1 mutants that cannot be cleaved by these proteases and examine the effect of the mutations on its antiviral function. Specific Aim 3 will identify the CREB3L1 target genes that inhibit HCV replication by microarray analysis. If these specific aims are achieved, we will have contributed novel information that will significantly enhance our understanding of the innate immune response. This new knowledge may reveal novel drug targets to treat HCV infection.