Manipulation of innate immunity by Polyomavirus T antigens

项目摘要 BKV是一种多瘤病毒，感染大多数人。BKV通常建立终身， 无症状持续感染偶尔，健康的人会排出少量的BKV颗粒， 尿，这一点，加上事实上，BKV生长在建立肾脏细胞系，导致了 认为病毒在泌尿道中持续存在。虽然在大多数情况下是无害的，但BKV可以进行生产 感染并诱导炎症反应，导致严重疾病，包括肾病， 免疫抑制患者的出血性膀胱炎。关于为什么BKV是热带的原因知之甚少。 肾及其向性是否仅限于泌尿系统。此外，决定 BKV生产性感染和持续性感染之间的平衡是未知的。 这个应用程序的重点是了解细胞反应的基础，导致生产性或持续性 感染BKV在原代人肾近曲小管上皮细胞（RPTE）中经历生产性感染， 其特征在于广泛的细胞死亡和释放约40个感染性颗粒/细胞。相比之下，BKV 在血管内皮细胞（VEC）中建立低水平的持续感染，引起最小的细胞病变， 在两个月的传代过程中，约10%的细胞表达病毒蛋白。RNA-seq显示 BKV在RPTE和BKV中诱导许多受E2 F家族转录因子调控的细胞周期基因， VEC。此外，许多干扰素刺激的基因（ISG）在BKV感染时上调， VEC但不是RPTE。 本研究将使用人类原代VEC培养物来鉴定影响感染的因素 成果：生产性或持久性。将单细胞转录组学应用于模拟和BKV感染的VEC 评估细胞亚群对感染的反应，并鉴定在限制细胞增殖中发挥作用的基因。 BKV复制。将使用以下两种方法评估VEC中BKV感染对干扰素应答的激活： 分子和功能（CRISPR敲除）方法，以确定途径的组成部分， 对限制病毒感染至关重要。特定的病毒突变体将被用来确定病毒的功能是什么 诱导干扰素应答所需的，以及在病毒感染的哪个阶段ISG是 诱导。点击化学将被用来确定哪些细胞因子调节之间的平衡 生产性和持续性感染。将使用多种方法的组合来鉴定细胞基因， 区分受限制的细胞和生产性感染的细胞。 这项工作将显着推进我们的知识BKV发病机制的特点，病毒的相互作用 与先天免疫系统，并通过确定细胞因子，促进或限制BKV感染， 细胞类型特异性的方式，并将提供有用的见解抗病毒剂的设计。

Project Summary BKV is a Polyomavirus that infects most of the human population. BKV typically establishes a lifelong, asymptomatic, persistent infection. Occasionally healthy humans excrete a small number of BKV particles in urine, and this, coupled with the fact that BKV grows productively in established kidney cell lines, has led to the view that the virus persists in the urinary tract. While harmless in most cases, BKV can undergo productive infection and induce an inflammatory response that results in serious diseases, including nephropathy and hemorrhagic cystitis in immunosuppressed patients. There is little knowledge as to why BKV is tropic for the kidney and whether its tropism is restricted to the urinary system. Furthermore, the factors governing the equilibrium between BKV productive infection and persistent infection are unknown. This application focuses on understanding the basis for cellular responses that lead to productive or persistent infection. BKV undergoes a productive infection in primary human renal proximal tubule epithelial cells (RPTE), characterized by extensive cell death and the release of about 40 infectious particles/cell. In contrast, BKV establishes a low-level persistent infection in vascular endothelial cells (VEC), causing minimal cytopathic effect with ~10% of the cells expressing viral proteins throughout two months of passaging. RNA-seq shows that BKV induces many cell cycle genes regulated by the E2F family of transcription factors in both RPTE and VEC. In addition, many interferon-stimulated genes (ISGs) are upregulated in response to BKV infection of VEC but not of RPTE. This research will use human primary human VEC cultures to identify the factors that influence infection outcomes: productive or persistent. Single cell transcriptomics will be applied to mock and BKV-infected VEC to assess the response of cell subpopulations to infection and to identify genes that play a role in restricting BKV replication. The activation of the interferon response by BKV infection in VEC will be assessed using both molecular and functional (CRISPR knockout) approaches to identify components of the pathway that are critical for limiting viral infection. Specific viral mutants will be used to determine what viral functions are required for the induction of the interferon response, as well as at which stage of the viral infection ISGs are induced. Click chemistry will be used to determine which cellular factors modulate the balance between productive and persistent infection. A combination of approaches will be used to identify cellular genes that distinguish restricted versus productively infected cells. This work will significantly advance our knowledge of BKV pathogenesis by characterizing the viral interaction with the innate immune system and by identifying cellular factors that promote or restrict BKV infection in a cell-type specific manner and will provide insights useful for the design of antiviral agents.