Mechanism and Inhibition of Thogotovirus Entry

在过去的十年中，新出现的病毒感染的发病率显著增加，这些病毒直接从动物传播给人类或由蚊子和蜱传播。人口和气候因素的变化可能是造成这一增长的原因。在美国，蜱是传播节肢动物传播疾病的主要媒介，包括可在人类中引起致命疾病的索戈托病毒、波旁病毒（BRBV）。与其他有包膜病毒一样，BRBV进入宿主细胞开始于与宿主细胞受体分子的附着，随后通过膜融合将病毒粒子的内容物递送到宿主细胞中。病毒附着和融合蛋白被抗体靶向，这些抗体有助于我们对病毒的天然免疫力，因此是经验证的治疗靶标。BRBV的单包膜糖蛋白（GP）负责与细胞受体的附着和催化融合。BRBV GP在结构上与水泡性口炎病毒的包膜糖蛋白G、单纯疱疹病毒的gB和杆状病毒的GP 64相关，这些被统称为III类融合原。我们对III类融合体及其抑制的理解落后于I类和II类病毒融合体，分别以流感HA和黄病毒E为例。我们已经开发了一套独特的工具和试剂，使我们能够表征现有的和新开发的抗BRBV GP单克隆抗体的结构和功能。我们将鉴定那些有效中和的抗体，鉴定它们的抑制机制，在小鼠疾病模型中测试中和和非中和抗体的功效，并鉴定这些抗体所起作用的相关thogotovirus的广度。使用嵌合VSV报告病毒，依赖于GP的BRBV的感染，我们将定义的确切步骤，在进入特定的抗体阻止感染。我们还将确定BRBV进入细胞的途径，并确定这一过程的宿主要求。在初步数据中，我们进行了CRISPR失活筛选，将葡糖神经酰胺合酶（UGCG）鉴定为BRBV进入哺乳动物细胞的重要宿主因子，并证明相关的thogotovirus也依赖于UGCG。使用遗传学方法的组合，加上药理学抑制和成像的单一病毒粒子在入境期间，我们将精确地描绘UGCG在入境的要求，并确定和表征在这个过程中增选的其他宿主因素。遗传和结构的研究将使我们能够映射GP的关键决定因素，需要在进入途径，这将协同与我们的抗体研究，以提供一个详细的进入和抑制机制的图片。拟议的研究的成功完成将提供新的见解的机制和结构要求的附件，内化和膜融合驱动的III类融合，揭示了宿主葡糖神经酰胺合酶的功能进入的机制，并确定抗体的目标GP阻断这些关键功能。

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.