Using Bacterial Effectors to Uncover Innate Immune Mechanisms Restricting Viral Replication in Bat Cells

利用细菌效应器揭示蝙蝠细胞中限制病毒复制的先天免疫机制

基本信息

批准号：
10592024
负责人：
Don Brad Gammon
金额：
$ 24.6万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-11 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10592024
关键词：
Address Affect Arbovirus Infections Arboviruses Attention Bacteria Bacterial Infections Bacteriophages Biological Assay Biological Models CRISPR/Cas technology Cell physiology Cells Cellular biology Chiroptera Cytoplasm Data Eukaryotic Cell Expression Library Family Filovirus Genes Glean Goals Grant Human Immune Immune Evasion Immune response Immune system Immunity Immunologic Factors Immunologics Immunology Immunosuppression Infection Innate Immune Response Insecta Integration Host Factors Libraries Methodology Methods Natural Immunity Pathogenesis Pathway interactions Population Predisposition Protein Secretion Proteins Proteomics Public Health RNA Interference RNA interference screen Research Rhabdoviridae Ring Finger Domain Shigella flexneri System Techniques Testing Therapeutic Togaviridae Vaccines Viral reservoir Virulence Factors Virus Virus Diseases Virus Replication Work Yeasts cell type comparative genomics genome editing genome-wide innate immune mechanisms innovation insight novel pathogen pathogenic bacteria pathogenic virus protein degradation protein function respiratory virus screening tool transcriptomic profiling transmission process ubiquitin ligase yeast two hybrid system

项目摘要

Project Summary Bats are important reservoirs for diverse viral pathogens affecting humans. However, we have a poor understanding of the key bat innate immunity factors that restrict virus replication. Functional assays that can identify bat factors that are truly relevant to combating viruses are needed to understand the innate immune mechanisms that ultimately define bat susceptibility to viral infection. While historically such functional screens have relied on genome-wide genomic editing (e.g. CRISPR-Cas9)- or RNA interference (RNAi)-based techniques, such platforms are unavailable for most bat species. Thus, new methods for uncovering functionally- relevant components of the bat immune response to virus infection are needed. To address this need, we have developed an innovative arbovirus "rescue" assay wherein immune evasion proteins (IEPs) encoded by mammalian pathogens can be expressed in bat cells and one can assay for changes in bat cell susceptibility to arbovirus infection. Enhancement of arbovirus replication after expression of a candidate IEP indicates that the IEP likely inhibits bat immunity mechanisms that normally restrict arbovirus replication. Using these IEPs as "tools", one can then identify the bat immunity factors these IEPs target. Thus, this screening methodology provides a mechanism to both identify novel IEPs and functionally-relevant components of the bat immune response. To discover IEPs that promote arbovirus replication in bat cells, we will screen an expression library encoding ~200 bacterial effector proteins. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that modulate or inhibit various eukaryotic cellular processes to promote bacterial replication. Many bacterial pathogens that replicate in the cytoplasm of eukaryotic host cells encode effectors that function as IEPs. Thus, we hypothesize that some effectors may suppress immune responses that restrict both bacteria and cytoplasmic viruses such as arboviruses. Indeed, our initial screens have identified four effectors that promote the replication of four different arboviruses when expressed in bat cells. We have characterized one of these effector screen "hits" as a novel ubiquitin ligase that targets an uncharacterized Ring Finger (RNF) Domain-containing protein for degradation in eukaryotic cells. Importantly, RNAi depletion of this RNF factor in human and bat cells promotes arbovirus replication, suggesting that it may be a novel component of human and bat immune responses. These results suggest that we can use bacterial effectors as tools to both inhibit, and identify, functionally-relevant immunity factors in bats. Our study has the following specific aims: 1) Identify bacterial effector proteins that promote arbovirus replication in bat cells;; 2) Identify bat proteins interacting with effector “hits” from our arbovirus rescue assays;; and 3) Determine which bat host factors interacting with effector protein hits affect viral replication. Our long-term goal is to use this model system to define the key bat innate immune mechanisms that restrict arbovirus replication.

项目概要蝙蝠是影响人类的多种病毒病原体的重要宿主，但我们的病毒病原体却很贫乏。了解限制病毒复制的关键蝙蝠先天免疫因素。需要确定与对抗病毒真正相关的蝙蝠因子，以了解先天免疫最终定义蝙蝠对病毒感染易感性的机制，而历史上是这样的功能筛选。依赖于基于全基因组的基因组编辑（例如 CRISPR-Cas9）或 RNA 干扰（RNAi）技术，这样的平台对于大多数蝙蝠物种来说是不可用的。因此，用于揭示功能的新方法为了满足这一需求，我们需要蝙蝠对病毒感染的免疫反应的相关成分。开发了一种创新的虫媒病毒“救援”测定法，同时编码的免疫逃避蛋白（IEP）哺乳动物病原体可以在蝙蝠细胞中表达，并且可以检测蝙蝠细胞对病毒的敏感性的变化表达候选 IEP 后虫媒病毒复制的增强表明 IEP 抑制通常可能限制虫媒病毒复制的蝙蝠免疫机制。有了“工具”，人们就可以识别这些 IEP 所针对的蝙蝠免疫因子，因此，这种筛选方法。提供了一种机制来识别新的 IEP 和蝙蝠免疫的功能相关成分为了发现促进蝙蝠细胞中虫媒病毒复制的 IEP，我们将筛选一个表达库。编码约 200 个细菌效应蛋白。细菌效应蛋白是由致病细菌分泌到体内的蛋白质。调节或抑制各种真核细胞过程以促进细菌生长的真核宿主细胞许多在真核宿主细胞的细胞质中复制的细菌病原体编码效应子。发挥 IEP 的作用。因此，我们假设某些效应器可能会抑制限制免疫反应事实上，我们的初步筛选已经确定了四种细菌和细胞质病毒。我们有在蝙蝠细胞中表达时促进四种不同虫媒病毒复制的效应子。将这些效应子筛选“命中”之一定性为一种新型泛素连接酶，靶向未定性的环 Finger (RNF) 含有结构域的蛋白质，可在真核细胞中降解。重要的是，RNAi 可以消除这种蛋白质人类和蝙蝠细胞中的 RNF 因子促进虫媒病毒复制，表明它可能是一种新成分这些结果表明我们可以使用细菌效应器作为两者的工具。抑制并鉴定蝙蝠中功能相关的免疫因子。我们的研究有以下具体目标：1) 鉴定促进蝙蝠细胞中虫媒病毒复制的细菌效应蛋白；2) 鉴定蝙蝠蛋白与我们的虫媒病毒救援试验中的效应子“命中”相互作用；以及 3) 确定哪些蝙蝠宿主因子与效应蛋白命中相互作用会影响病毒复制。定义限制虫媒病毒复制的关键蝙蝠先天免疫机制。