Mammalian innate immune response to virus infection is meditated by many cell-intrinsic pathways. RNA viruses, such as Sendai virus, which replicate in the cytoplasm, trigger the RIG-I-like receptor pathway, which activates the transcription factor, IRF3. Activated IRF3 translocates to the nucleus and induces transcription of the genes which encode interferons, the major antiviral cytokines. Interestingly, IRF3 activates another interferon-independent antiviral pathway, called RIG-I induced pathway of apoptosis (RIPA). For activating RIPA, IRF3 translocates from the cytoplasm to mitochondria. RIPA requires linear polyubiquitination of IRF3 by the enzyme complex, LUBAC; ubiquitinated IRF3 binds to Bax and translocates it to mitochondria causing the release of Cytochrome C, activation of caspases and apoptosis of the infected cell. Here, we report that Otulin, the deubiquitinase that removes linear polyubiquitin chains, inhibits RIPA by deubiquitinating IRF3. Ablation of Otulin expression enhanced RIPA and its overexpression inhibited RIPA. In virus-infected cells, to overcome Otulin-mediated inhibition, RIPA actively degrades Otulin. This degradation required sequential actions of RIPA-activated Caspase 3 and proteasomes. Caspase 3 cleaved Otulin at D31; the D31A mutant was not cleaved at all. The caspase-cleaved fragment was totally degraded by proteasomes, which was preceded by its K48-linked ubiquitination. Mass spectrometric analysis of Otulin identified K64 and K197 as the ubiquitinated residues. Otulin interacted with LUBAC after virus infection and the E3-ubiquitin ligase, HOIP, a component of LUBAC, ubiquitinated Otulin to trigger its proteasome-mediated degradation. To assess the impact of Otulin degradation on RIPA-mediated antiviral action, we expressed, in Otulin-ablated cells, a non-degradable mutant of Otulin, in which D31, K64 and K197 had been mutated. The cells expressing the Otulin mutant were less susceptible to virus-induced apoptosis, because RIPA was less active, and consequently virus replication was more robust. Thus, our study has revealed an important positive feedback loop of RIPA.

哺乳动物对病毒感染的先天免疫反应由许多细胞内在途径调节。仙台病毒等 RNA 病毒在细胞质中复制，触发 RIG-I 样受体通路，激活转录因子 IRF3。激活的 IRF3 易位至细胞核并诱导编码干扰素（主要抗病毒细胞因子）的基因转录。有趣的是，IRF3 激活了另一种不依赖干扰素的抗病毒途径，称为 RIG-I 诱导细胞凋亡途径 (RIPA)。为了激活 RIPA，IRF3 从细胞质转移到线粒体。RIPA 需要通过酶复合物 LUBAC 对 IRF3 进行线性多聚泛素化；泛素化的 IRF3 与 Bax 结合并将其转移到线粒体，导致细胞色素 C 的释放、半胱天冬酶的激活和受感染细胞的凋亡。这里，我们报告 Otulin，一种去除线性多聚泛素链的去泛素化酶，通过去泛素化 IRF3 来抑制 RIPA。Otulin 表达的消融增强了 RIPA，其过表达抑制了 RIPA。在病毒感染的细胞中，为了克服 Otulin 介导的抑制，RIPA 主动降解 Otulin。这种降解需要 RIPA 激活的半胱天冬酶 3 和蛋白酶体的连续作用。Caspase 3 在 D31 处切割 Otulin；D31A 突变体根本没有被切割。半胱天冬酶切割的片段被蛋白酶体完全降解，之前是其 K48 连接的泛素化。Otulin 的质谱分析将 K64 和 K197 鉴定为泛素化残基。Otulin 在病毒感染后与 LUBAC 相互作用，E3-泛素连接酶 HOIP 是 LUBAC 的一个组成部分，泛素化 Otulin 以触发其蛋白酶体介导的降解。为了评估 Otulin 降解对 RIPA 介导的抗病毒作用的影响，我们在去除 Otulin 的细胞中表达了一种不可降解的 Otulin 突变体，其中 D31、K64 和 K197 发生了突变。表达 Otulin 突变体的细胞不易受病毒诱导的细胞凋亡的影响，因为 RIPA 活性较低，因此病毒复制更加稳健。因此，我们的研究揭示了 RIPA 的重要正反馈回路。