Molecular Control of IFITM3 in Restricting Influenza Virus Infection

 描述（由申请人提供）：流感病毒感染及其并发症仍然是美国死亡的主要原因，每年给国家带来高达870亿美元的经济负担。我们和其他人已经证明干扰素诱导的跨膜蛋白3（IFITM 3）在体外抑制流感病毒感染，并且IFITM 3也已经被证明对于小鼠和人类中对流感病毒感染的先天抗性是必需的。尽管IFITM 3在广泛抑制流感和其他病毒方面具有重要意义，但这一知识尚未激发新的治疗方法，因为仍不清楚IFITM 3如何阻碍病毒感染以及IFITM 3的运输和功能如何受到生化调节。我们的总体假设是IFITM 3的稳定性和定位是由一个 一系列翻译后修饰，确保IFITM 3正确递送和锚定到内溶酶体，其中其膜内结构域降低膜流动性以防止流感病毒融合/感染。目的1是基于我们的新发现，即E3泛素连接酶NEDD 4是IFITM 3细胞水平和活性的负调节剂。NEDD 4可能代表了一种新的药物靶点，用于改善IFITM 3介导的针对流感病毒的先天免疫，我们的工作研究了NEDD 4对IFITM 3的泛素化，以及这种修饰和其他修饰之间的串扰将提供关于IFITM 3调节机制的分子细节，这些机制必须被理解，以便最终设计出基于IFITM 3生物学的治疗方法。目的2是建立在我们的发现，即一个特定的DHHC结构域的棕榈酰转移酶能够棕榈酰IFITM 3，我们以前证明这是一个必要的修改IFITM 3抗病毒活性。我们将分析潜在的IFITM 3棕榈酰化缺陷所造成的多态性报告这种酶，从而奠定了基础，其研究作为流感病毒的易感因素。我们的初步数据还表明，IFITM 3棕榈酰化必须发生在特定的细胞区室，以观察其活性的积极影响。因此，我们将调查假定DHHC本地化机制，占这种效果。这一系列实验将确定关键的DHHC运输和特异性决定因素，并揭示哪些细胞隔室支持IFITM 3的适当棕榈酰化/活化。在目标3中，我们将检查IFITM 3膜内区域在改变细胞膜以抑制病毒感染中的功能。我们提供的证据表明，IFITM 3的预测跨膜结构域作为膜内结构域。膜内结构域经常改变膜结构，我们的初步数据表明IFITM 3确实降低了膜流动性。因此，我们将研究IFITM 3膜内结构域在降低膜流动性和抑制病毒感染中的必要性和充分性。在所有这些研究的过程中，我们将应用所获得的知识来开发最佳活性但最小尺寸的抗病毒多肽构建体。总体而言，这些研究目标应该揭示控制和利用IFITM 3对抗流感病毒和病毒性疾病的分子策略。

 DESCRIPTION (provided by applicant): Influenza virus infection and its complications remains a leading cause of death in the US, and burdens the nation economically with costs of up to $87 billion annually. We, and others, have shown that the interferon- induced transmembrane protein 3 (IFITM3) inhibits influenza virus infections in vitro, and IFITM3 has also been shown to be essential for innate resistance to influenza virus infection in both mice and humans. Despite the importance of IFITM3 in broadly inhibiting influenza and other viruses, this knowledge has not yet inspired new therapeutics because it remains unclear precisely how IFITM3 hinders viral infection and how IFITM3 trafficking and function are regulated biochemically. Our overall hypothesis is that IFITM3 stability and localization are controlled by a series of post-translational modifications that ensure proper delivery and anchoring of IFITM3 to endolysosomes, where its intramembrane domains decrease membrane fluidity to prevent influenza virus fusion/infection. Aim 1 is based on our novel discovery that the E3 ubiquitin ligase NEDD4 is a negative regulator of IFITM3 cellular levels and activity. NEDD4 may represent a novel drug target for improv- ing IFITM3-mediated innate immunity against influenza virus, and our work examining IFITM3 ubiquitination by NEDD4, and the crosstalk between this and other modifications will provide molecular details regarding IFITM3 regulatory mechanisms that must be understood in order to ultimately devise such therapeutics based on IFITM3 biology. Aim 2 is built upon our discovery that a specific DHHC domain-containing palmitoyltransferase is able to palmitoylate IFITM3, which we previously demonstrated is an essential modification necessary for IFITM3 antiviral activity. We will analyze potential IFITM3 palmitoylation defects caused by polymorphisms reported for this enzyme, thus laying the groundwork for its study as an influenza virus susceptibility factor. Our preliminary data also suggest that IFITM3 palmitoylation must occur at a specific cellular compartment for positive effects on its activity to be observed. Thus we will investigate putative DHHC localization mechanisms that account for this effect. This series of experiments will identify critical DHHC trafficking and specificity determinants, and reveal which cellular compartments support the proper palmitoylation/activation of IFITM3. In Aim 3 we will examine the function of IFITM3 intramembrane regions in altering cellular membranes to inhibit virus infections. We have provided evidence that IFITM3's predicted transmembrane domains act instead as intramembrane domains. Intramembrane domains often alter membrane structure, and our preliminary data indicates that IFITM3 indeed decreases membrane fluidity. Thus, we will examine the necessity and sufficiency of IFITM3 intramembrane domains in decreasing membrane fluidity and inhibiting virus infection. In the course of all of these studies we will appy the knowledge gained toward the development of an optimally active but minimally sized antiviral poly- peptide construct. Overall, these research aims should reveal molecular strategies for controlling and exploiting IFITM3 in fighting influenza virus and viral diseases.