Embry Aaron, Schad David, Rex Emily A, Alto Neal M, Gammon Don B
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
bioRxiv. 2024 Nov 4:2024.11.04.621956. doi: 10.1101/2024.11.04.621956.
Arboviruses are a group of arthropod-transmitted viruses that pose a significant threat to public health. Identifying host factors that inhibit arbovirus infection is critical for the development of strategies to prevent or treat these infections. Previously, we showed that bacterial effector proteins can be used as molecular tools to identify host immunity factors in insect cells that restrict arbovirus replication (Embry et al., 2024). Bacteria secrete effectors into the host cell cytoplasm to inhibit various innate immune defenses. Here, we apply our bacterial effector screening system to identify host antiviral immunity factors in two mammalian hosts - bats and humans. By screening a library of 210 effectors encoded by seven distinct bacterial pathogens, we identified three bacterial effectors (IpaH4, SopB, and SidM) that enhance the replication of both togaviruses and rhabdoviruses in bat and human cells. We also discovered several effectors that enhance arbovirus replication in a virus- or host-specific manner. We further characterize the mechanism by which the encoded E3 ubiquitin ligase, IpaH4, enhances arbovirus infection in mammalian cells. Using yeast two-hybrid, ubiquitin-activated interaction traps, ubiquitination assays and cellular approaches, we show the uncharacterized mammalian RING-domain containing protein, RNF214, to be directly targeted by IpaH4 for ubiquitination-mediated degradation. Phylogenetic analyses of RNF214 proteins indicate they are widely conserved among many vertebrate species, suggesting an important evolutionary function. We show that RNF214 overexpression suppresses arbovirus infections in a manner dependent upon its putative E3 ubiquitin ligase activity, while RNF214 depletion enhances these infections in human and bat cells. These data suggest that RNF214 proteins are important innate immune factors involved in combating viral infection. Collectively, our work shows that bacterial effectors can be useful tools for uncovering novel mammalian antiviral machinery.
虫媒病毒是一类由节肢动物传播的病毒，对公共卫生构成重大威胁。识别抑制虫媒病毒感染的宿主因子对于制定预防或治疗这些感染的策略至关重要。此前，我们表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（Embry等人，2024年）。细菌将效应蛋白分泌到宿主细胞质中，以抑制各种先天免疫防御。在此，我们应用我们的细菌效应蛋白筛选系统，在两种哺乳动物宿主——蝙蝠和人类中识别宿主抗病毒免疫因子。通过筛选由七种不同细菌病原体编码的210种效应蛋白文库，我们鉴定出三种细菌效应蛋白（IpaH4、SopB和SidM），它们可增强披膜病毒和弹状病毒在蝙蝠和人类细胞中的复制。我们还发现了几种以病毒或宿主特异性方式增强虫媒病毒复制的效应蛋白。我们进一步表征了编码的E3泛素连接酶IpaH4增强哺乳动物细胞中虫媒病毒感染的机制。使用酵母双杂交、泛素激活相互作用陷阱、泛素化测定和细胞方法，我们表明未表征的含RING结构域的哺乳动物蛋白RNF214被IpaH4直接靶向，进行泛素化介导的降解。RNF214蛋白的系统发育分析表明，它们在许多脊椎动物物种中广泛保守，表明其具有重要的进化功能。我们表明，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的缺失则增强了人类和蝙蝠细胞中的这些感染。这些数据表明，RNF214蛋白是参与对抗病毒感染的重要先天免疫因子。总体而言，我们的工作表明细菌效应蛋白可成为揭示新型哺乳动物抗病毒机制的有用工具。
