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

项目摘要 尽管病毒感染继续对人类健康造成重大负担， 并且对于许多病毒性病原体，疫苗仍然不可用。更好地理解这些机制， 免疫系统感知和控制病毒感染的能力将使新的策略的发展成为可能， 他们的预防和治疗。干扰素（IFN）是对病毒感染的免疫应答的关键介质， 但过量会导致慢性炎症和自身免疫。因此，IFN的产生是 严格监管。复制病毒产生RNA，RNA传感器RIG-I和 MDA5。RIG-I检测病毒如仙台病毒（SeV）和水泡性口炎病毒（VSV），而MDA5检测病毒如仙台病毒（SeV）和水泡性口炎病毒（VSV）。 对于IFN诱导以控制小核糖核酸病毒如泰勒氏鼠脑脊髓炎病毒（TMEV）是必需的， 和杯状病毒属，鼠诺如病毒（MNoV）。一旦被激活，这些传感器就会发出一个共同的信号， 导致IFN转录诱导并建立抗病毒环境的级联反应。虽然 线性遍在蛋白链组装复合物（LUBAC）先前已显示抑制IFN 通过RIG-I诱导，我们的研究表明LUBAC亚基HOIL 1对于IFN诱导是必需的， MDA5在鼠树突状细胞和成纤维细胞中的表达，并在体内控制MNoV感染。而且我们 已经发现HOIL 1的E3泛素连接酶活性对于TMEV感染期间的IFN诱导是必需的， 成纤维细胞然而，HOIL 1 E3连接酶活性调节MDA5依赖性IFN-γ的机制是不确定的。 诱导，以及HOIL 1泛素化的生物学后果是未知的。我们假设HOIL 1 和LUBAC被选择性地募集到MDA5信号通路，其中HOIL 1催化 该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HOIL 1 E3连接酶突变体中MDA5信号级联中阻断的位置。 细胞，并确定其他LUBAC蛋白结构域，调节MDA5信号和IFN诱导。二是 将使用候选人和无偏见的生化方法来确定HOIL 1相互作用和泛素化 MDA5信号传导过程中的蛋白质。最后，我们将测试HOIL 1 E3连接酶活性是否差异调节IFN-γ， 在MNoV、SeV、VSV和TMEV感染小鼠过程中体内诱导和病毒发病机理，并将使用细胞 型特异性敲除小鼠，以鉴定需要HOIL 1诱导IFN和控制MNoV的细胞类型 体内持续感染。我们希望我们的研究能够确定一种新的IFN调节机制，从而 揭示了病毒和IFN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.