Mechanism and Inhibition of Thogotovirus Entry

托戈托病毒侵入机制及抑制

• 批准号: 10568571
• 负责人: Adrianus CM Boon
• 金额: $ 69.58万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-22 至 2027-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10568571
• 关键词: Acids; Affinity; Amblyomma; Ambylomma americanum; Animals; Antibodies; Antibody Repertoire; Antiviral Agents; Area; B-Lymphocytes; Baculoviruses; Binding; Biochemical; Biological Assay; Bourbon virus; CRISPR-mediated transcriptional activation; Cell fusion; Cell membrane; Cell-Matrix Junction; Cells; Ceramide glucosyltransferase; Chimeric Proteins; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; County; Coupled; Cryoelectron Microscopy; Data; Demographic Factors; Disease; Disease model; Epitopes; Escape Mutant; Flavivirus; Future; Genetic; Glycoproteins; Glycosphingolipids; Hand; Human; Image; Imaging Device; Immunity; Immunize; Incidence; Individual; Infection; Influenza Hemagglutinin; Integration Host Factors; Kansas; Kinetics; Knock-out; Life Cycle Stages; Mammalian Cell; Maps; Mediating; Membrane Fusion; Membrane Glycoproteins; Midwestern United States; Missouri; Molecular Cloning; Molecular Conformation; Monoclonal Antibodies; Mus; Natural Immunity; Null Lymphocytes; Pathway interactions; Patients; Process; Productivity; Proteins; Rabies virus; Reagent; Receptor Cell; Reporter; Resistance; Rhabdoviridae; Role; Serum; Simplexvirus; Site; Sorting; Structure; Testing; Thogotovirus; Ticks; United States; Vesicular stomatitis Indiana virus; Viral; Viral Envelope Proteins; Viral Fusion Proteins; Viral Physiology; Virion; Virus; Virus Diseases; arboviral disease; biophysical techniques; burden of illness; climatic factors; cohort; cross reactivity; efficacy testing; env Gene Products; experimental study; genetic approach; genetic information; glycoprotein G; group competition; human disease; human pathogen; inhibiting antibody; insight; mouse model; mutant; neutralizing antibody; particle; pharmacologic; protective efficacy; protein function; receptor; response; success; synergism; therapeutic target; tick transmission; tool; transmission process; unpublished works; uptake; vaccine development; vector mosquito; vector transmission; viral RNA

The past decade has seen a significant increase in the incidence of emerging virus infections that transmit directly from animals to humans or are vectored by mosquitos and ticks. Changes in demographics and climatic factors likely contribute to this increase. In the United States, ticks are the dominant vector for transmitting arthropod borne diseases including the Thogotovirus, Bourbon virus (BRBV) which can cause fatal disease in humans. Like other enveloped viruses, entry of BRBV begins with attachment to host-cell receptor molecules with subsequent membrane fusion to deliver the contents of the virion into the host cell. Viral attachment and fusion proteins are targeted by antibodies that contribute to our natural immunity against viruses and are consequently a validated therapeutic target. The single envelope glycoprotein (GP) of BRBV is responsible for both attachment to cellular receptors and catalyzing fusion. BRBV GP is structurally related to the envelope glycoprotein G of vesicular stomatitis virus, gB of herpes simplex virus, and GP64 of baculovirus which collectively are termed class III fusogens. Our understanding of class III fusogens and their inhibition lags behind the class I and II viral fusogens, exemplified by influenza HA, and flavivirus E respectively. We have developed a set of unique tools and reagents that will allow us to characterize existing and newly developed monoclonal antibodies against BRBV GP both structurally and functionally. We will identify those that are potently neutralizing, identify their mechanism of inhibition, test the efficacy of neutralizing and non-neutralizing antibodies in a mouse model of disease, and identify the breadth of related thogotoviruses against which such antibodies function. Using chimeric VSV reporter viruses that depend on the GP of BRBV for infection, we will define the precise step in entry by which specific antibodies impede infection. We will also determine the entry pathway of BRBV into cells and define the host requirements for this process. In preliminary data, we carried out a CRISPR inactivation screen that identified glucosylceramide synthase (UGCG) as an important host factor for entry of BRBV into mammalian cells and demonstrate related thogotoviruses are also dependent upon UGCG. Using a combination of genetic approaches, coupled with pharmacological inhibition and imaging of single virions during entry, we will precisely delineate the requirement for UGCG in entry, and identify and characterize additional host factors coopted during this process. Genetic and structural studies will permit us to map the critical determinants on GP required to coopt host-factors during the entry pathway, which will synergize with our antibody studies to provide a detailed mechanistic picture of entry and its inhibition. Successful completion of the proposed studies will provide new insights into the mechanism and structural requirements for attachment, internalization and membrane fusion driven by a class III fusogen, uncover the mechanism by which the host glucosylceramide synthase functions in entry, and identify antibodies that target GP to block those critical functions.

在过去的十年中，新出现的病毒感染的发生率显着增加，这些病毒感染直接从动物传播到人类，或者被蚊子和壁虱载入。人口统计学和气候因素的变化可能会导致这种增加。在美国，壁虱是传播节肢动物传播疾病的主要载体，包括thogotovirus，波旁病毒（BRBV），可能引起人类致命疾病。像其他包围病毒一样，BRBV的进入始于与宿主细胞受体分子的附着，随后膜融合，将病毒粒子的含量输送到宿主细胞中。病毒附着和融合蛋白是由有助于我们对病毒的天然免疫力的抗体靶向的，因此是经过验证的治疗靶标。 BRBV的单个包膜糖蛋白（GP）既是对细胞受体的附着和催化融合的原因。 BRBV GP在结构上与囊泡口腔炎病毒的包膜糖蛋白G，单纯疱疹病毒的GB和baculovirus的GP64相关，这些GP64集体称为III类融合剂。我们对III类融合剂及其抑制作用的理解滞后于I级和II级病毒融合剂，分别以流感HA和Flavivivirus e为例。我们已经开发了一套独特的工具和试剂，使我们能够在结构和功能上表征针对BRBV GP的现有和新开发的单克隆抗体。我们将确定那些有力中和，确定其抑制作用的机制，测试中和和非中和抗体在小鼠疾病模型中的疗效，并确定这种抗体功能的相关Thogotovires的广度。使用依赖于BRBV的GP进行感染的嵌合VSV报道病毒，我们将定义进入特定抗体会阻碍感染的进入的确切步骤。我们还将确定BRBV进入单元格的进入途径，并定义此过程的宿主要求。在初步数据中，我们进行了CRISPR灭活筛查，该筛查将葡萄糖基酶合酶（UGCG）确定为将BRBV进入哺乳动物细胞的重要宿主因子，并证明相关的THOGOTOVIRES也取决于UGCG。使用遗传方法的组合，再加上药理学抑制和对单个病毒体的成像，我们将精确地描述进入入口时对UGCG的需求，并确定和表征此过程中使用的其他宿主因素。遗传和结构研究将使我们能够在入口途径期间对Cookt宿主因子所需的GP的关键决定因素绘制，这将与我们的抗体研究协同作用，以提供详细的入学机理及其抑制作用。拟议研究的成功完成将为通过III类菌根驱动的附着，内在化和膜融合的机制和结构要求提供新的见解，发现宿主葡萄糖基酶合酶在入口中功能的机制，并识别靶向GP以阻止这些关键功能的抗体。