Regulated Intramembrane Proteolysis of CREB3L1 in Innate Antiviral Response

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染了世界3%的人口，并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。针对丙型肝炎病毒蛋白的药物很难开发，因为丙型肝炎病毒复制过程中的高突变率使耐药病毒株迅速出现。目前丙型肝炎病毒感染的治疗是以干扰素为基础的，干扰素是一种针对RNA病毒感染的先天性免疫反应。然而，这种治疗只有部分有效。因此，了解其他先天抗病毒反应可能会揭示治疗丙型肝炎病毒感染亟需的新策略。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系中的丙型肝炎病毒感染方面发挥了重要作用。该途径由cAMP反应元件结合蛋白3样蛋白1(CREB3L1)介导，其功能尚不清楚。CREB3L1属于转录因子家族，作为插入内质网(ER)的膜结合前体被合成，并被称为调节膜内蛋白分解(RIP)的过程激活。根据我们目前对RIP的理解，我们认为丙型肝炎病毒在内质网中的复制导致CREB3L1被位点1蛋白酶(S1P)和位点2蛋白酶(S2P)切割。蛋白水解性切割使CREB3L1的NH2末端片段从膜上释放出来，并进入细胞核，激活其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进行检验。具体目标1将确定丙型肝炎病毒复制刺激CREB3L1裂解的机制。我们将研究由丙型肝炎病毒编码膜蛋白的表达诱导的内质网应激是否触发CREB3L1的切割。具体目标2将确定其抗病毒功能是否需要S1P和S2P催化的CREB3L1裂解。主要的方法是使CREB3L1突变不能被这些酶切割，并检查突变对其抗病毒功能的影响。特异靶3将通过微阵列分析确定抑制丙型肝炎病毒复制的CREB3L1靶基因。如果实现了这些特定的目标，我们将提供新的信息，这些信息将显著增强我们对先天性免疫反应的理解。这一新知识可能会揭示治疗丙型肝炎病毒感染的新药物靶点。 公共卫生相关性：该项目旨在研究一种由一种名为CREB3L1的细胞蛋白介导的新的抗病毒反应，该蛋白能有效地抑制丙型肝炎病毒(HCV)的复制。全球有1.7亿人感染丙型肝炎病毒，这种病毒感染是美国肝功能衰竭的主要原因。目前以干扰素为基础的治疗丙型肝炎病毒感染只具有部分感染性。因此，了解CREB3L1介导的明显不依赖干扰素的抗病毒途径，可能会揭示迫切需要的治疗丙型肝炎病毒感染的新策略。

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. PUBLIC HEALTH RELEVANCE: This project is aimed to study a novel antiviral response mediated by a cellular protein named CREB3L1 that potently inhibits hepatitis C virus (HCV) replication. HCV infects 170 million people worldwide, and the viral infection is the leading cause of liver failure in the United States. The current interferon-based treatment for HCV infection is only partially infective. Thus, understanding the CREB3L1-mediated antiviral pathway, which apparently is interferon-independent, may reveal much needed new strategies to treat HCV infection.