Infectious cell entry pathway of human-infecting thogotoviruses

感染人类的托戈托病毒的感染细胞进入途径

基本信息

批准号：
10373971
负责人：
Paul William Rothlauf
金额：
$ 3.45万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10373971
关键词：
Aerosols Affect Anabolism Arthropod Vectors Back Baculoviruses Biochemical Biological Biological Assay Bourbon virus CRISPR screen Cell Culture Techniques Cell Line Cell membrane Cells Ceramide glucosyltransferase Cessation of life Chemicals Chimera organism Chiroptera Culicidae Detection Dhori virus Disease Diversity Library Ebola Endogenous Retroviruses Event Family Foundations Genes Genetic Genome Geography Glycoproteins Glycosphingolipids Goals Heartland virus Herpes Simplex Infections Human Infection Integration Host Factors Knock-out Knowledge Laboratories Lentivirus Lujo virus Maps Mediating Membrane Fusion Membrane Proteins Molecular Cloning Molecular Conformation Orthomyxoviridae Pathway interactions Persons Phenotype Proteins RNA Viruses Rabies Reagent Reporter Resistance to infection Role Severe Fever with Thrombocytopenia Syndrome Virus Structure Technology Testing Therapeutic Therapeutic Intervention Thogoto virus Thogotovirus Vaccines Variant Vesicular stomatitis Indiana virus Viral Virion Virus Virus Diseases arthropod-borne combat env Gene Products experimental study follow-up genome wide screen genome-wide human disease imaging approach infection risk influenzavirus inhibitor insight mutant new therapeutic target novel pathogen prevent recombinant virus response restoration tick-borne tissue tropism tool transmission process vacuolar H+-ATPase vector tick virology virus envelope

项目摘要

Project Summary/Abstract Viruses in the genus Orthomyxoviridae and family Thogotovirus are globally widespread pathogens capable of causing significant and fatal human disease. These enveloped, segmented, negative-sense RNA viruses are mainly transmitted to hosts by a tick vector, though there is some evidence for transmission by mosquitoes and aerosols. Thogoto, Dhori, and Bourbon virus infections in humans have caused significant disease and, in some cases, death. There are no vaccines or therapeutics to prevent or alleviate thogotovirus infections, as most steps in the viral lifecycle of this entire family of viruses are poorly understood. Defining the lifecycle of thogotoviruses is the first step towards developing treatments to combat thogotovirus infections. Thogotoviruses use a single viral glycoprotein to mediate entry into host cells. While these glycoproteins have been structurally defined as class III viral fusogens and shown to undergo conformational changes in response to low pH, their role in entry is otherwise unknown. I have successfully replaced the single glycoprotein of eGFP-expressing vesicular stomatitis virus (VSV) with the glycoproteins of Bourbon, Dhori, and Thogoto virus in order to generate biosafety level 2, replication-competent, reporter viruses. With these novel tools, I have initially confirmed a role for pH in the Bourbon virus entry pathway and have conducted a genome-wide CRISPR-Cas9 screen, which has revealed the requirement for a host protein, glucosylceramide synthase (UGCG), in viral entry. The underlying hypothesis for my proposal is that virus-specific host factors dictate thogotovirus entry into cells. I will examine this hypothesis in two specific aims. In specific aim 1, I will 1) perform additional, more robust CRISPR-Cas9 screens, and 2) determine the role of identified host factors in thogotovirus entry. In specific aim 2, I will use genetic, chemical, and imaging approaches to define the role of UGCG in thogotovirus entry. Completion of these studies will reveal the entry pathways and host factors required for thogotovirus entry into cells and will define the mechanistic role of UGCG in the entry pathway of these viruses. Consequently, these studies will provide insight into the basic virology of an understudied family of human-infecting viruses.

项目概要/摘要正粘病毒科和托戈托病毒科的病毒是全球广泛传播的病原体，能够造成严重和致命的人类疾病。这些有包膜、分段、负义 RNA 病毒是主要通过蜱虫媒介传播给宿主，尽管有一些证据表明通过蚊子和气溶胶。 Thogoto、Dhori 和 Bourbon 病毒感染人类已引起严重疾病，并且有些情况下，死亡。没有疫苗或治疗方法可以预防或减轻托戈托病毒感染，因为人们对整个病毒家族的病毒生命周期的大多数步骤知之甚少。定义生命周期 thogotoviruses 是开发对抗 thogotovirus 感染治疗方法的第一步。索戈托病毒使用单一病毒糖蛋白介导进入宿主细胞。虽然这些糖蛋白具有在结构上被定义为 III 类病毒融合剂，并显示出响应时会发生构象变化对于低 pH 值，它们在进入中的作用尚不清楚。我已经成功替换了单糖蛋白表达 eGFP 的水泡性口炎病毒 (VSV)，具有波旁病毒、多里病毒和托戈托病毒的糖蛋白以生成生物安全 2 级、具有复制能力的报告病毒。有了这些新颖的工具，我最初确认了 pH 在波本病毒进入途径中的作用，并进行了全基因组研究 CRISPR-Cas9 筛选，揭示了对宿主蛋白葡萄糖神经酰胺合酶的需求（UGCG），病毒进入。我的提议的基本假设是病毒特异性宿主因素决定索戈托病毒进入细胞。我将从两个具体目标来检验这个假设。在具体目标 1 中，我将 1) 进行额外的、更强大的 CRISPR-Cas9 筛选，以及 2) 确定已识别宿主的作用托戈托病毒进入的因素。在具体目标 2 中，我将使用遗传、化学和成像方法来定义 UGCG 在 thogotovirus 进入中的作用。完成这些研究将揭示进入途径和宿主 thogotovirus 进入细胞所需的因素，并将定义 UGCG 在进入细胞中的机制作用这些病毒的传播途径。因此，这些研究将深入了解病毒的基本病毒学尚未研究的人类感染病毒家族。