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

• 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
• 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

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.

项目摘要/摘要 Orthomyxoviridae属和家族Thogotovirus属的病毒是全球广泛的病原体 引起严重和致命的人类疾病。这些包裹，分段，负敏感的RNA病毒是 尽管有一些证据表明，蚊子和 气溶胶。人类的Thogoto，Dhori和波旁病毒感染已引起严重的疾病，并在 有些情况，死亡。没有疫苗或治疗剂可以预防或减轻thogotovirus感染，例如 整个病毒家族的病毒生命周期中的大多数步骤都了解不足。定义生命周期 Thogotoviruses是开发治疗方法以对抗Thogotovirus感染的第一步。 Thogotovirus使用单个病毒糖蛋白介导进入宿主细胞的进入。这些糖蛋白有 在结构上被定义为III类病毒融合剂，并显示出对响应的构象变化 对于低pH值，它们在进入中的作用是未知的。我成功地取代了 表达EGFP的囊泡口腔炎病毒（VSV），带有波旁威士忌，dhori和Thogoto病毒的糖蛋白 为了生成生物安全2级，复制能力的报道病毒。有了这些新颖的工具，我有 最初证实了pH在波旁病毒进入途径中的作用，并进行了全基因组的作用 CRISPR-CAS9屏幕，揭示了宿主蛋白，葡萄糖基酶合酶的要求 （UGCG），在病毒进入中。我提出的基本假设是病毒特异性宿主因素 指示Thogotovirus进入细胞。我将以两个具体的目的研究这一假设。在特定的目标1中，我 会1）执行额外，更健壮的CRISPR-CAS9屏幕，2）确定已识别主机的作用 Thogotovirus进入的因素。在特定的目标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。这些研究的完成将揭示进入途径和主机 Thogotovirus进入细胞所需的因素，并将定义UGCG的机械作用 这些病毒的途径。因此，这些研究将提供对一个的基本病毒学的见解 研究的人类感染病毒家族。