Infectious cell entry pathway of human-infecting thogotoviruses

项目摘要/摘要 正粘病毒科和ThogotoVirus属病毒是全球分布广泛的病原体，能够 导致重大和致命的人类疾病。这些包膜的、分段的、负义的RNA病毒是 主要通过扁虱媒介传播给宿主，但也有一些证据表明是通过蚊子和 气雾剂。Thogoto、Dhori和Bourbon病毒在人类中的感染已导致重大疾病和 在某些情况下，死亡。目前还没有疫苗或疗法来预防或减轻托戈病毒感染，因为 这整个病毒家族的病毒生命周期中的大多数步骤都鲜为人知。定义的生命周期 托戈托病毒是开发抗托戈托病毒感染的治疗方法的第一步。 甲状旁腺病毒使用单一的病毒糖蛋白来调节进入宿主细胞。而这些糖蛋白有 在结构上被定义为III类病毒融合原，并被证明经历了构象变化的反应 对于低pH值，它们在进入过程中的作用是未知的。我已经成功地取代了单一的糖蛋白 用Bourbon、Dhori和Thogoto病毒的糖蛋白表达EGFP的水泡性口炎病毒(VSV) 以产生生物安全级别2、复制能力强的报告病毒。有了这些新奇的工具，我有了 初步确认了pH在波旁病毒进入途径中的作用，并进行了全基因组 CRISPR-Cas9筛选，揭示了对宿主蛋白葡萄糖神经酰胺合成酶的需求 (UGCG)，在病毒进入中。我的提议的基本假设是病毒特异性宿主因素 指示索戈托病毒进入细胞。我将从两个具体目标来检验这一假设。在具体目标1中，我 是否将1)执行更强大的额外CRISPR-CAS9筛查，以及2)确定已确定的主机的角色 索戈托病毒进入的因素。在具体目标2中，我将使用遗传、化学和成像方法来定义 UGCG在弓形虫病毒进入中的作用。这些研究的完成将揭示进入途径和宿主 梭状病毒进入细胞所需的因子，并将确定UGCG在进入细胞中的机械作用 这些病毒的途径。因此，这些研究将提供对基础病毒学的洞察。 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.