The posttranslational modification (PTM) of innate immune sensor proteins by ubiquitin or ubiquitin-like proteins is crucial for regulating antiviral host responses. The cytoplasmic dsRNA receptor melanoma differentiation-associated protein 5 (MDA5) undergoes several PTMs including ISGylation within its first caspase activation and recruitment domain (CARD), which promotes MDA5 signaling. However, the relevance of MDA5 ISGylation for antiviral immunity in an infected organism has been elusive. Here, we generated knock-in mice (MDA5 K23R/K43R ) in which the two major ISGylation sites, K23 and K43, in MDA5, were mutated. Primary cells derived from MDA5 K23R/K43R mice exhibited abrogated endogenous MDA5 ISGylation and an impaired ability of MDA5 to form oligomeric assemblies, leading to blunted cytokine responses to MDA5 RNA-agonist stimulation or infection with encephalomyocarditis virus (EMCV) or West Nile virus. Phenocopying MDA5 −/− mice, the MDA5 K23R/K43R mice infected with EMCV displayed increased myocardial injury and mortality, elevated viral titers, and an ablated induction of cytokines and chemokines compared to WT mice. Molecular studies identified human HERC5 (and its functional murine homolog HERC6) as the primary E3 ligases responsible for MDA5 ISGylation and activation. Taken together, these findings establish the importance of CARD ISGylation for MDA5-mediated RNA virus restriction, promoting potential avenues for immunomodulatory drug design for antiviral or anti-inflammatory applications.

中文翻译：

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MDA5 ISGylation 对于免疫信号转导控制病毒复制和发病机制至关重要


泛素或泛素样蛋白对先天免疫传感器蛋白的翻译后修饰 （PTM） 对于调节抗病毒宿主反应至关重要。细胞质 dsRNA 受体黑色素瘤分化相关蛋白 5 （MDA5） 在其第一个半胱天冬酶激活和募集结构域 （CARD） 内经历多个 PTM，包括 ISGylation，从而促进 MDA5 信号传导。然而，MDA5 ISGylation 与感染生物体中抗病毒免疫的相关性一直难以捉摸。在这里，我们生成了敲入小鼠 （MDA5 K23R/K43R），其中 MDA5 中的两个主要 ISGylation 位点 K23 和 K43 发生了突变。来源于 MDA5 K23R/K43R 小鼠的原代细胞表现出内源性 MDA5 ISGylation 被消除和 MDA5 形成寡聚组装体的能力受损，导致细胞因子对 MDA5 RNA 激动剂刺激或脑心肌炎病毒 （EMCV） 或西尼罗河病毒感染的反应减弱。表型复制 MDA5 −/− 小鼠，与 WT 小鼠相比，感染 EMCV 的 MDA5 K23R/K43R 小鼠表现出心肌损伤和死亡率增加、病毒滴度升高以及细胞因子和趋化因子的消融诱导。分子研究确定人 HERC5（及其功能性小鼠同源物 HERC6）是负责 MDA5 ISGylation 和激活的主要 E3 连接酶。综上所述，这些发现确立了 CARD ISGylation 对 MDA5 介导的 RNA 病毒限制的重要性，为免疫调节药物设计提供了用于抗病毒或抗炎应用的潜在途径。