Key resources and reagents.

Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.

虫媒病毒（Arbovirus）是一类多样的、经昆虫传播的病原体，给全球公共卫生带来严峻挑战。鉴定在不同真核宿主中对抗虫媒病毒复制的进化保守宿主因子至关重要，此类因子可调控病毒产能性复制与流产性复制之间的平衡，进而决定病毒的宿主范围。本研究利用我们在鳞翅目（Lepidoptera）昆虫细胞中发现的天然流产性虫媒病毒感染模型，并借助细菌效应蛋白（bacterial effector protein）来挖掘限制虫媒病毒复制的宿主因子。细菌效应蛋白是病原细菌分泌至真核宿主细胞内、可抑制宿主抗菌防御系统的蛋白质。由于细菌与病毒可能遭遇共通的宿主防御机制，我们推测部分细菌效应蛋白或许能够抑制鳞翅目昆虫细胞中限制虫媒病毒复制的宿主因子。因此，我们以细菌效应蛋白作为分子工具，在鳞翅目昆虫细胞中筛选限制四种不同虫媒病毒复制的宿主因子。通过对7种不同病原菌编码的210种效应蛋白进行筛选，我们鉴定出数种可单独恢复四种虫媒病毒复制的效应蛋白。我们证实，这些效应蛋白具备多样的酶促活性，而此类活性正是打破虫媒病毒宿主限制所必需的。我们进一步对弗氏志贺菌（Shigella flexneri）编码的IpaH4进行表征，发现其作为E3泛素连接酶（E3 ubiquitin ligase），可直接对两种进化保守蛋白SHOC2和PSMC1进行泛素化修饰，促进二者在昆虫与人类细胞内的降解。我们证实，在昆虫或人类细胞中敲低SHOC2或PSMC1均可促进虫媒病毒复制，表明二者是在无脊椎动物与脊椎动物宿主中均保守的古老病毒限制因子。综上，本研究揭示了一种全新的病原体导向策略，可用于鉴定保守的抗菌机制、新的效应蛋白功能，以及SHOC2与PSMC1在病毒限制中的保守作用。