Infectious cell entry pathway of human-infecting thogotoviruses

项目总结/摘要 正粘病毒科（Orthomyxoviridae）和索戈托病毒科（Thogotovirus）中的病毒是全球广泛分布的病原体，其能够 导致严重和致命的人类疾病。这些有包膜、分节、反义RNA病毒是 主要通过蜱媒传播给宿主，尽管有一些证据表明通过蚊子传播， 气溶胶Thogoto，Dhori和Bourbon病毒感染在人类中引起了严重的疾病， 有些情况下，死亡。没有疫苗或治疗剂来预防或减轻thogotovirus感染， 对这整个病毒家族的病毒生命周期中的大多数步骤知之甚少。定义生命周期 thogotoviruses是朝着开发对抗thogotovirus感染的治疗方法迈出的第一步。 索戈托病毒使用单个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类病毒融合子，并显示在应答中发生构象变化 到低pH值，它们在进入中的作用是未知的。我已经成功地替换了 具有波旁、多里和托戈托病毒的糖蛋白的表达eGFP的水泡性口炎病毒（VSV） 以产生生物安全等级2、复制能力、报告病毒。有了这些新奇的工具， 最初证实了pH在波旁病毒进入途径中的作用，并进行了全基因组研究。 CRISPR-Cas9筛选，揭示了对宿主蛋白葡糖神经酰胺合酶的需求 （UGCG），在病毒进入中。我的建议的基本假设是，病毒特异性宿主因素 指示thogotovirus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， 将1）进行额外的，更强大的CRISPR-Cas9筛选，和2）确定鉴定的宿主的作用 Thogotovirus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。这些研究的完成将揭示进入途径和宿主 这些因子是thogotovirus进入细胞所需的，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，这些研究将提供深入了解的基础病毒学的一个 未被充分研究的人类感染病毒家族。

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.