Embry Aaron, Schad David F, Rex Emily A, Alto Neal M, Gammon Don B
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
PLoS Pathog. 2025 Apr 22;21(4):e1013035. doi: 10.1371/journal.ppat.1013035. eCollection 2025 Apr.
Arboviruses are arthropod-borne viruses that pose significant threats to human and animal health. Previously, we demonstrated that bacterial effector proteins can serve as molecular tools to identify host immunity factors in insect cells that restrict arbovirus replication. In this study, we apply our bacterial effector screening system to identify immunity factors in two mammalian hosts-bats and humans. Our screens identified three bacterial effectors (IpaH4, SopB and SidM) that enhanced the replication of unrelated arboviruses in bat and human cells. We also discovered several effectors that enhanced arbovirus replication in an arbovirus- or host-specific manner. Focusing on the Shigella flexneri-encoded E3 ubiquitin ligase IpaH4, we identified the uncharacterized mammalian really interesting new gene (RING)-domain-containing protein RNF214 as a direct target that is ubiquitinated and degraded by IpaH4. RNF214 belongs to a large family of RING finger (RNF) proteins that primarily function as E3 ubiquitin ligases and that have diverse roles in regulating and mediating innate immune responses to disparate pathogens. Phylogenetic analyses reveal that RNF214 is highly conserved across vertebrate species, suggesting a conserved role in host defense. Functional studies demonstrate that RNF214 overexpression suppresses arbovirus infection in a manner dependent on its putative E3 ubiquitin ligase activity, whereas RNF214 depletion enhances viral replication in both human and bat cells. Furthermore, knockout of RNF214 did not alter the upregulation of interferon (IFN)-stimulated gene expression during infection or upon treatment of cells with IFN. Screening of 11 RNA and DNA viruses, revealed that RNF214 specifically restricts single-stranded RNA (ssRNA) viruses. These findings establish RNF214 as a critical component of the innate immune response against ssRNA viruses that may function independently of the IFN response. More broadly, our work highlights the utility of bacterial effector proteins as powerful tools for uncovering novel antiviral machinery in mammals.
虫媒病毒是由节肢动物传播的病毒，对人类和动物健康构成重大威胁。此前，我们证明细菌效应蛋白可作为分子工具，用于识别昆虫细胞中限制虫媒病毒复制的宿主免疫因子。在本研究中，我们应用细菌效应筛选系统来识别两种哺乳动物宿主（蝙蝠和人类）中的免疫因子。我们的筛选鉴定出三种细菌效应蛋白（IpaH4、SopB和SidM），它们能增强无关虫媒病毒在蝙蝠和人类细胞中的复制。我们还发现了几种以虫媒病毒或宿主特异性方式增强虫媒病毒复制的效应蛋白。聚焦于弗氏志贺菌编码的E3泛素连接酶IpaH4，我们鉴定出未表征的含哺乳动物真核生物中有趣新基因（RING）结构域的蛋白RNF214为IpaH4泛素化并降解的直接靶点。RNF214属于RING指蛋白（RNF）大家族，主要作为E3泛素连接酶发挥作用，在调节和介导对不同病原体的固有免疫反应中具有多种作用。系统发育分析表明，RNF214在脊椎动物物种中高度保守，提示其在宿主防御中具有保守作用。功能研究表明，RNF214的过表达以依赖其假定的E3泛素连接酶活性的方式抑制虫媒病毒感染，而RNF214的缺失则增强人类和蝙蝠细胞中的病毒复制。此外，RNF214基因敲除并未改变感染期间或用干扰素处理细胞后干扰素（IFN）刺激基因表达的上调。对11种RNA和DNA病毒的筛选显示，RNF214特异性限制单链RNA（ssRNA）病毒。这些发现确立了RNF214作为针对ssRNA病毒的固有免疫反应的关键组成部分，其可能独立于IFN反应发挥作用。更广泛地说，我们的工作突出了细菌效应蛋白作为揭示哺乳动物新型抗病毒机制的强大工具的实用性。
