Mechanism and Inhibition of Thogotovirus Entry

在过去十年中，直接从动物传播给人类或由蚊子和蜱虫传播的新出现的病毒感染的发病率显著增加。人口和气候因素的变化可能促成这一增长。在美国，蜱虫是传播节肢动物传播疾病的主要媒介，包括可导致人类致命疾病的波旁病毒（BRBV）。与其他包膜病毒一样，BRBV的进入首先与宿主细胞受体分子结合，随后进行膜融合，将病毒粒子的内容物运送到宿主细胞。病毒附着和融合蛋白是抗体的目标，有助于我们对病毒的天然免疫，因此是一个有效的治疗靶点。BRBV的单包膜糖蛋白（GP）既与细胞受体结合又催化融合。BRBV GP在结构上与水疱性口炎病毒的包膜糖蛋白G、单纯疱疹病毒的包膜糖蛋白gB和杆状病毒的包膜糖蛋白GP64相关，它们统称为III类融合原。我们对III类融合原及其抑制作用的了解落后于I类和II类病毒融合原，例如流感HA和黄病毒E。我们已经开发了一套独特的工具和试剂，使我们能够从结构和功能上表征现有的和新开发的抗BRBV GP单克隆抗体。我们将确定那些有效中和的，确定它们的抑制机制，在小鼠疾病模型中测试中和和非中和抗体的功效，并确定这些抗体对相关thogotovirus的作用范围。使用依赖BRBV的GP进行感染的嵌合VSV报告病毒，我们将确定特异性抗体阻止感染的准确进入步骤。我们还将确定BRBV进入细胞的途径，并确定宿主对这一过程的需求。在初步数据中，我们进行了CRISPR失活筛选，发现葡萄糖神经酰胺合成酶（UGCG）是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。结合遗传学方法，结合进入过程中单个病毒粒子的药理抑制和成像，我们将精确地描述进入过程中对UGCG的需求，并确定和表征在此过程中采用的其他宿主因子。遗传和结构研究将使我们能够绘制出GP在进入途径中吸收宿主因子所需的关键决定因素，这将与我们的抗体研究协同作用，提供进入及其抑制的详细机制图。这些研究的成功完成将为III类融合原驱动的附着、内化和膜融合的机制和结构要求提供新的见解，揭示宿主葡萄糖神经酰胺合酶在进入过程中的作用机制，并确定靶向GP阻断这些关键功能的抗体。

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.