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

项目总结 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 而且许多病毒病原体仍然没有疫苗可用。通过以下方式对机制有更深入的了解 免疫系统感知和控制病毒感染将使开发新的策略成为可能 他们的预防和治疗。干扰素是对病毒感染的免疫反应的关键介质， 但过量的摄入会导致慢性炎症和自身免疫。因此，干扰素的生产 受到严格监管。复制的病毒产生的RNA在细胞内被RNA传感器、RIG-I和 MDA5.RIG-I检测到仙台病毒(SeV)和水疱性口炎病毒(VSV)等病毒，而MDA5是 对于诱导干扰素以控制小核糖核酸病毒如泰勒氏小鼠脑脊髓炎病毒(TMEV)是必不可少的， 和杯状病毒，小鼠诺如病毒(MNoV)。一旦被激活，这些传感器就会启动一个共同的信号 导致干扰素转录诱导并建立抗病毒环境的级联反应。虽然 线性泛素链组装复合体(LUBAC)先前已被证明能抑制干扰素 在RIG-I的诱导下，我们的研究表明，LUBAC亚基HOIL1是干扰素诱导所必需的 MDA5在小鼠树突状细胞和成纤维细胞中的表达，并在体内控制MNoV的感染。此外，我们 已发现HOIL1的E3泛素连接酶活性是TMEV感染过程中诱导干扰素所必需的 成纤维细胞。然而，HOIL1 E3连接酶活性调节MDA5依赖的干扰素的机制 诱导，HOIL1泛素化的生物学后果尚不清楚。我们假设霍伊尔1 和LUBAC被选择性地招募到MDA5信号通路中，其中HOIL1催化泛素化 途径中的一个或多个信号分子，以促进信号转导和干扰素基因的转录。 首先，我们将确定HOIL1 E3连接酶突变体中MDA5信号级联的阻断位置 细胞，并确定其他调控MDA5信号和干扰素诱导的LUBAC蛋白结构域。第二，我们 将使用候选和无偏生化方法来鉴定HOIL1相互作用因子和泛素化 MDA5信号传导过程中的蛋白质。最后，我们将测试HOIL1 E3连接酶活性是否对干扰素有不同的调节作用 MNoV、SeV、VSV和TMEV感染小鼠的体内诱导和病毒致病机制，并将使用细胞 特定类型的敲除小鼠识别需要HOIL1来诱导干扰素和控制MNOV的细胞类型 体内持续感染。我们期望我们的研究能够发现一种新的干扰素调节机制，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.