The Functions of IFITM Proteins in Control of Influenza A Virus Infection

IFITM 蛋白在控制甲型流感病毒感染中的功能

• 批准号: 8951589
• 负责人: I-Chueh Huang
• 金额: $ 41.13万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8951589
• 关键词: Functions; IFITM; Proteins; Control; Influenza

DESCRIPTION (provided by applicant): Influenza A virus (IAV) is an important pathogen. It is responsible for 200,000 hospitalizations and 41,000 deaths in the United States annually. Our previous studies identified that interferon-inducible transmembrane (IFITM) proteins are critical factors for the immune control of IAV in vitro and in vivo. We demonstrated that, uniquely among known viral restriction factors, IFITM proteins restricted viruses before they entered cells. Our overall goal of this proposed project is to understand comprehensively the functions of IFITM proteins and the mechanisms by which these proteins restrict IAV infection. These studies consist of three specific aims. Aim 1. We will determine whether the stability of IFITM3 polymorphisms contribute to their differential control of IAV. We have demonstrated that IFITM3 expression is closely regulated by secretory carrier membrane protein 3 (SCAMP3). We have also noticed that the stability of a recently identified polymorphic IFITM3 ( 21 IFITM3) may explain its correlation to the poor clinical prognosis. In this aim, a series of immunoprecipitatio, immunoblotting, drug inhibitory, and viral entry assays will be performed to determine the pathway contributing to IFITM3 degradation and the mechanism by which SCAMP3 stabilizes its expression. We will also investigate the role of SCAMP3 in the differential control of IAV by wild-type IFITM3 and 21 IFITM3. Aim 2. We will investigate whether IFITM proteins alter intracellular cholesterol homeostasis, resulting in viral entry restriction. In our preliminary studies, we observed that IFITM3 interferes with the homeostasis of intracellular cholesterol via its interaction with vesicle-associated membrane protein-associated protein A (VAPA). In this aim we will clarify whether IFITM-mediated cholesterol accumulation contributes to viral entry restriction. To do so, we will comprehensively characterize the properties of wild-type and functional negative IFITM3 variants and their effects on viral entry under a normal or a cholesterol-depleted condition. We will also evaluate whether this cholesterol accumulation can be similarly induced by IFN. Finally, the effects of cholesterol accumulation on fusion between virion and cell membranes will be determined by confocal imaging. Aim 3. We will examine whether IFITM proteins interfere with the late stage of endocytosis, thereby blocking the progression of virion trafficking. Because IFITM3 also interacts with several vesicle trafficking-related proteins such as CD63, alteration of endosomal trafficking is another potential mechanism of IFITM-mediated restriction. In the aim, we will perform live-cell imaging and cell-free organelle fusion assays to examine whether IFITM proteins interfere with the late stage of endocytosis. The effects of IFITM3 on the composition of endosomal phosphatidylinositol and on the interactions between CD63 and several cellular factors critical for endosomal trafficking (e.g., phosphatidylinositol 4-kinases (PI4K) and adaptor protein 3 (AP-3)) will also be characterized by confocal microscopy and biochemical analyses. We will further evaluate whether PI4K, AP-3, and CD63 alter IFITM- mediated viral entry restriction.

描述（申请人提供）：甲型流感病毒（IAV）是一种重要的病原体。在美国，每年有20万人住院治疗，4.1万人死亡。我们之前的研究发现干扰素诱导跨膜（IFITM）蛋白是体外和体内IAV免疫控制的关键因素。我们证明，在已知的病毒限制因子中，IFITM蛋白在病毒进入细胞之前限制了病毒。我们的总体目标是全面了解IFITM蛋白的功能以及这些蛋白限制IAV感染的机制。这些研究包括三个具体目标。目的1。我们将确定IFITM3多态性的稳定性是否有助于它们对IAV的差异控制。我们已经证明IFITM3的表达受到分泌载体膜蛋白3 （SCAMP3）的密切调节。我们还注意到，最近发现的多态IFITM3 （21 IFITM3）的稳定性可以解释其与不良临床预后的相关性。为此，将进行一系列免疫沉淀、免疫印迹、药物抑制和病毒进入试验，以确定IFITM3降解的途径以及SCAMP3稳定其表达的机制。我们还将研究SCAMP3在野生型IFITM3和21型IFITM3对IAV的差异控制中的作用。目标2。我们将研究IFITM蛋白是否改变细胞内胆固醇稳态，导致病毒进入限制。在我们的初步研究中，我们观察到IFITM3通过与囊泡相关膜蛋白相关蛋白A （VAPA）的相互作用干扰细胞内胆固醇的稳态。在这个目的中，我们将澄清ifitm介导的胆固醇积累是否有助于限制病毒进入。为此，我们将全面表征野生型和功能性阴性IFITM3变异的特性，以及它们在正常或胆固醇耗尽条件下对病毒进入的影响。我们还将评估是否这种胆固醇积累可以类似地由IFN诱导。最后，胆固醇积累对病毒粒子与细胞膜融合的影响将通过共聚焦成像来确定。目标3。我们将研究IFITM蛋白是否干扰内吞作用的后期，从而阻断病毒粒子运输的进展。由于IFITM3还与几种囊泡运输相关的蛋白（如CD63）相互作用，因此内体运输的改变是ifitm介导的限制的另一种潜在机制。为此，我们将进行活细胞成像和无细胞细胞器融合试验，以检查IFITM蛋白是否干扰后期内吞作用。IFITM3对内体磷脂酰肌醇组成的影响，以及CD63与内体运输关键细胞因子（如磷脂酰肌醇4-激酶（PI4K）和衔接蛋白3 (AP-3)）之间的相互作用，也将通过共聚焦显微镜和生化分析来表征。我们将进一步评估PI4K、AP-3和CD63是否会改变IFITM介导的病毒进入限制。