在RNA病毒感染过程中，病原模式识别受体（PRRs）会在细胞内发生迁移和再定位，靠近病毒的复制场所，识别病毒RNA。下游信号蛋白也会发生迁移，在空间上接近PRRs，以便于传递感染信号。然而，PRRs和信号蛋白的迁移和亚细胞定位，尚不清楚。 我们前期研究发现，新城疫病毒NDV感染HeLa细胞和DF-1细胞，能够诱导形成稳定的应激颗粒（SG），促进干扰素的产生。通过免疫荧光观察，发现一部分病毒mRNA、PRRs (PKR、RIG-I、MDA5、TLR3)、 以及大量的免疫信号蛋白在NDV感染过程中发生迁移，聚集于SG，揭示SG有可能是天然免疫系统识别病毒感染、发生信号传递的一个枢纽。本项目拟通过亲和沉淀分离NDV诱导的SG，进行质谱分析和Western blot，并通过免疫荧光实验，鉴定和验证SG的免疫信号蛋白组；探讨信号蛋白聚集于SG的生物学意义；调查以上结果是否普遍存在于RNA病毒感染；探究泛素化修饰是否参与信号蛋白的SG聚集。

During RNA virus infection, Pathogen Pattern Recognition Receptors (PRRs) would migrate to the virus replication site, thereby sensing virus RNA. The downstream signaling proteins would also migrate and aggregate to PRRs, transmitting the infection signaling to transcription factor. However, the migration and subcellular location of PRRs and signaling proteins in response to virus infection are unclear. Our study shows that Newcastle disease virus (NDV) infection activates PKR and induces the stable formation of stress granule (SG) in HeLa cells and DF-1 cells, promoting interferon production. Immunofluoresence shows that a certain amount of viral mRNA and PRRs (PKR, RIG-I, MDA5, TLR3) aggregate into foci and co-localize with SG; meanwhile, lots of immune signaling proteins migrate to SG, revealing that SG is a potential center for virus recognition and signaling transduction. In this proposal, we aim to investigate SG as a center of immune signaling initiation and transmission. First, we will perform the mass spectrometric analysis using NDV-induced SG, which will be purified by pulling down via a tag on SG core protein. The identified SG immune proteome will be validated with Western blot and Immunofluoresence. Next, we will investigate whether the SG aggregation of signaling proteins promotes signaling recognition and transmission, interferon (IFN) production. Furthermore, above results will be tested in the case of poly (I:C) or other RNA virus stimulation. Finally, we will check whether ubiquitination is involved in the SG migration and aggregation of signal proteins.