Defining the differential roles of HOIL1 and the Linear Ubiquitin Chain Assembly Complex in interferon induction by MDA5 and RIG-I during viral infection.

项目概要 尽管病毒感染继续给人类健康带来巨大负担，但有效的治疗方法 许多病毒病原体仍然无法获得疫苗。对机制有更深入的了解 免疫系统感知和控制病毒感染的能力将有助于开发新的策略 他们的预防和治疗。干扰素 (IFN) 是病毒感染免疫反应的关键介质， 但过量会导致慢性炎症和自身免疫。因此 IFN 的产生是 受到严格监管。复制病毒产生的 RNA 可以在细胞内被 RNA 传感器、RIG-I 和 丙二醛5。 RIG-I 检测仙台病毒 (SeV) 和水疱性口炎病毒 (VSV) 等病毒，而 MDA5 则检测病毒 对于诱导 IFN 控制小核糖核酸病毒（如泰勒氏鼠脑脊髓炎病毒 (TMEV)）至关重要， 以及杯状病毒、鼠诺如病毒（MNoV）。一旦激活，这些传感器就会启动通用信号 级联反应导致 IFN 的转录诱导并建立抗病毒环境。虽然 线性泛素链组装复合物 (LUBAC) 先前已被证明可以抑制 IFN RIG-I 诱导，我们的研究表明 LUBAC 亚基 HOIL1 对于 IFN 诱导至关重要 MDA5存在于鼠树突状细胞和成纤维细胞中，并控制体内MNoV感染。此外，我们 发现 HOIL1 的 E3 泛素连接酶活性对于 TMEV 感染期间的 IFN 诱导至关重要 成纤维细胞。然而，HOIL1 E3 连接酶活性调节 MDA5 依赖性 IFN 的机制 HOIL1 泛素化的生物学后果尚不清楚。我们假设 HOIL1 和 LUBAC 被选择性地招募到 MDA5 信号通路，其中 HOIL1 催化 途径中的一种或多种信号分子以促进IFN基因的信号转导和转录。 首先，我们将确定 HOIL1 E3 连接酶突变体中 MDA5 信号级联中阻断的位置 细胞，并鉴定调节 MDA5 信号传导和 IFN 诱导的其他 LUBAC 蛋白结构域。第二，我们 将使用候选和无偏见的生化方法来识别 HOIL1 相互作用子和泛素化 MDA5 信号转导过程中的蛋白质。最后，我们将测试 HOIL1 E3 连接酶活性是否差异调节 IFN 小鼠 MNoV、SeV、VSV 和 TMEV 感染期间的体内诱导和病毒发病机制，并将使用细胞 类型特异性敲除小鼠，以确定需要 HOIL1 诱导 IFN 和控制 MNoV 的细胞类型 体内持续感染。我们期望我们的研究能够确定干扰素调节的新机制，从而 揭示病毒和干扰素介导的自身免疫性疾病的潜在治疗靶点。

PROJECT SUMMARY Despite the significant burden that viral infections continue to impose on human health, effective therapeutics and vaccines are still unavailable for many viral pathogens. A stronger understanding of the mechanisms by which the immune system senses and controls viral infections will enable the development of new strategies for their prevention and treatment. Interferons (IFN) are critical mediators of the immune response to viral infections, but excessive amounts can lead to chronic inflammation and autoimmunity. The production of IFN is therefore tightly regulated. Replicating viruses produce RNAs that are detected within cells by RNA sensors, RIG-I and MDA5. RIG-I detects viruses such as Sendai virus (SeV) and vesicular stomatitis virus (VSV), whereas MDA5 is essential for IFN induction to control picornaviruses such as Theiler’s murine encephalomyelitis virus (TMEV), and the Calicivirus, murine norovirus (MNoV). Once activated, these sensors initiate a common signaling cascade that leads to the transcriptional induction of IFN and establishes an anti-viral environment. Although the Linear Ubiquitin Chain Assembly Complex (LUBAC) has been shown previously to inhibit IFN induction by RIG-I, our studies have shown that LUBAC subunit, HOIL1, is essential for IFN induction by MDA5 in murine dendritic cells and fibroblasts, and to control MNoV infection in vivo. Furthermore, we have found that the E3 ubiquitin ligase activity of HOIL1 is essential for IFN induction during TMEV infection of fibroblasts. However, the mechanism by which HOIL1 E3 ligase activity regulates MDA5-dependent IFN induction, and the biological consequences of ubiquitination by HOIL1 are unknown. We hypothesize that HOIL1 and LUBAC are recruited selectively to the MDA5 signaling pathway, wherein HOIL1 catalyzes ubiquitination of one or more signaling molecules in the pathway to facilitate signal transduction and transcription of IFN genes. First, we will determine the location of the blockade in the MDA5 signaling cascade in HOIL1 E3 ligase mutant cells, and identify other LUBAC protein domains that regulate MDA5 signaling and IFN induction. Second, we will use both candidate and unbiased biochemical approaches to identify HOIL1-interactors and ubiquitinated proteins during MDA5 signaling. Last, we will test whether HOIL1 E3 ligase activity differentially regulates IFN induction and viral pathogenesis in vivo during MNoV, SeV, VSV and TMEV infection of mice, and will use cell type-specific knock-out mice to identify the cell types that require HOIL1 to induce IFN and control MNoV persistent infection in vivo. We expect our studies to identify a novel mechanism of IFN regulation, thereby revealing potential therapeutic targets for viral and IFN-mediated autoimmune diseases.