Department of Microbiology and Molecular Genetics, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Box 9102, Southborough, MA 01772-9102, USA.
J Virol. 2010 Apr;84(7):3220-9. doi: 10.1128/JVI.02241-09. Epub 2010 Jan 13.
Recognition of invading viruses by the host is elicited by cellular sensors which trigger signaling cascades that lead to type I interferon (IFN) gene expression. Retinoic acid-inducible gene I (RIG-I) has emerged as a key receptor for the detection of viral RNA in the cytosol, inducing IFN-mediated innate immune responses to limit viral replication through its interaction with MAVS (also called IPS-1, CARDIF, or VISA). Upon the recognition of viral RNA, the Lys-172 residue of RIG-I undergoes ubiquitination induced by tripartite motif protein 25 (TRIM25), an essential protein for antiviral signal transduction. Here we demonstrate that phosphorylation represents another regulatory mechanism for RIG-I-mediated antiviral activity. Using protein purification and mass spectrometry analysis, we identified three phosphorylation sites in the amino-terminal caspase recruitment domains (CARDs) of RIG-I. One of these residues, Thr-170, is located in close proximity to Lys-172, and we speculated that its phosphorylation may affect Lys-172 ubiquitination and functional activation of RIG-I. Indeed, a RIG-I mutant carrying a phosphomimetic Glu residue in place of Thr-170 loses TRIM25 binding, Lys-172 ubiquitination, MAVS binding, and downstream signaling ability. This suggests that phosphorylation of RIG-I at Thr-170 inhibits RIG-I-mediated antiviral signal transduction. Immunoblot analysis with a phospho-specific antibody showed that the phosphorylation of the RIG-I Thr-170 residue is present under normal conditions but rapidly declines upon viral infection. Our results indicate that Thr-170 phosphorylation and TRIM25-mediated Lys-172 ubiquitination of RIG-I functionally antagonize each other. While Thr-170 phosphorylation keeps RIG-I latent, Lys-172 ubiquitination enables RIG-I to form a stable complex with MAVS, thereby inducing IFN signal transduction.
宿主通过细胞传感器识别入侵的病毒，这些传感器触发信号级联反应，导致 I 型干扰素（IFN）基因表达。视黄酸诱导基因 I（RIG-I）已成为细胞质中检测病毒 RNA 的关键受体，通过与 MAVS（也称为 IPS-1、CARDIF 或 VISA）相互作用，诱导 IFN 介导的固有免疫反应，限制病毒复制。在识别病毒 RNA 后，RIG-I 的赖氨酸 172 残基通过三部分基序蛋白 25（TRIM25）发生泛素化，TRIM25 是抗病毒信号转导的必需蛋白。在这里，我们证明磷酸化是 RIG-I 介导的抗病毒活性的另一种调节机制。通过蛋白质纯化和质谱分析，我们在 RIG-I 的氨基末端半胱天冬酶募集结构域（CARD）中鉴定出三个磷酸化位点。其中一个残基 Thr-170 位于靠近赖氨酸 172 的位置，我们推测其磷酸化可能影响赖氨酸 172 的泛素化和 RIG-I 的功能激活。事实上，携带 Thr-170 磷酸化模拟 Glu 残基的 RIG-I 突变体丧失了与 TRIM25 的结合、赖氨酸 172 的泛素化、MAVS 的结合和下游信号转导能力。这表明 RIG-I 在 Thr-170 位点的磷酸化抑制了 RIG-I 介导的抗病毒信号转导。用磷酸特异性抗体进行的免疫印迹分析表明，在正常条件下，RIG-I Thr-170 残基存在磷酸化，但在病毒感染后迅速下降。我们的结果表明，RIG-I 的 Thr-170 磷酸化和 TRIM25 介导的赖氨酸 172 泛素化在功能上相互拮抗。虽然 Thr-170 磷酸化使 RIG-I 潜伏，但赖氨酸 172 泛素化使 RIG-I 能够与 MAVS 形成稳定的复合物，从而诱导 IFN 信号转导。
