Function of ISG15 during Viral Infection

描述（由申请方提供）：干扰素（IFN）通过诱导数百种干扰素刺激基因（ISG）产物发挥抗病毒和免疫调节作用。虽然已经研究了其中的几个基因，如PKR，RNaseL和Mx，但数百个这些基因仍然没有已知的功能。这是一个建议，以确定这些未知基因之一，干扰素刺激基因15（ISG 15）的生理重要性和作用机制。ISG15是IFN诱导的蛋白质，其含有两个泛素样结构域。除了与IFN刺激的细胞中的蛋白质偶联之外，ISG15还从IFN处理的细胞中释放并在IFN处理的患者的血清中发现。释放的ISG15充当触发自然杀伤（NK）细胞增殖和增强的杀伤、来自PBMC的IFNg分泌和树突状细胞（DC）分化的细胞因子。我们已经发现ISG15在体内具有抗病毒作用，并且ISG15在组织（脾和肝）中的表达由病毒感染诱导。重要的是，我们在急性病毒感染期间的血清中发现了ISG15，这与作为细胞因子的作用一致。总之，这些数据使我们假设ISG15是一种细胞因子，其在IFN应答期间起作用以调节DC和NK细胞的功能，这对于先天性和潜在的适应性免疫应答至关重要。本提案的目的是通过三个目标来检验这一基本假设。目的1中的研究旨在评估ISG15是否在体内发挥重要的免疫或抗病毒作用。特别是，我们将完成ISG15基因敲除小鼠的产生，并确定这些小鼠是否对病毒感染具有改变的抵抗力。目的2中的研究旨在确定ISG15是否作为细胞外细胞因子调节免疫应答。我们将确定从感染小鼠血清中分离的ISG15的生化性质，确定其抗病毒活性所需的ISG15的结构域，并建立ISG15细胞因子功能的体外测定。目的3中的研究旨在鉴定ISG15受体。从这些研究中获得的结果应该为ISG15的功能意义提供进一步的见解，并提供其发挥抗病毒活性的潜在机制。

DESCRIPTION (provided by applicant): Interferons (IFN) exert anti-viral and immunomodulatory effects through their induction of hundreds of interferon stimulated gene (ISGs) products. While several of these genes, such as PKR, RNaseL, and Mx have been studied, hundreds of these genes still have no known function. This is a proposal to define the physiologic importance and mechanisms of action of one of these unknown genes, IFN stimulated gene 15 (ISG15). ISG15 is an IFN induced protein that contains two ubiquitin like domains. In addition to coupling to proteins in IFN stimulated cells, ISG15 is released from IFN treated cells and found in the serum of IFN treated patients. Released ISG15 acts as a cytokine that triggers natural killer (NK) cell proliferation and enhanced killing, IFNg secretion from PBMC, and dendritic cell (DC) differentiation. We have found that ISG15 has antiviral effects in vivo, and that ISG15 expression in tissues (spleen and liver) is induced by virus infection. Importantly, we have found ISG15 in the serum during acute virus infection, consistent with a role as a cytokine. Together these data lead us to the hypothesis that ISG15 is a cytokine that acts during IFN responses to modulate the function of DCs and NK cells that are critical for innate and potentially adaptive immune responses. The goal of this proposal is to test this underlying hypothesis via three Aims. Studies in Aim 1 are designed to evaluate if ISG15 plays an important immunologic or anti-viral role in vivo. In particular, we will complete the generation of an ISG15 knockout mouse and determine if these mice have altered resistance to viral infection. The studies in Aim 2 are designed to determine if ISG15 functions as an extracellular cytokine to regulate immune responses. We will determine the biochemical nature of ISG15 isolated from sera of infected mice, determine the structural domains of ISG15 required for its anti-viral activity, and establish an in vitro assay for ISG15 cytokine function. The studies in Aim 3 are designed to identify the ISG15 receptor. The results obtained from these studies should provide further insight into the functional significance of ISG15, and provide a potential mechanism by which it exerts its antiviral activity.