Manipulation of innate immunity by Polyomavirus T antigens

项目摘要 BKV是一种多瘤病毒，感染大多数人类人口。BKV通常会建立一种终身的， 无症状的持续性感染。偶尔健康的人会排出少量的BKV颗粒 尿液，再加上BKV在已建立的肾脏细胞系中高效生长的事实，导致了 认为病毒持续存在于尿路。虽然在大多数情况下BKV是无害的，但BKV可以在 感染并引发炎症反应，导致严重疾病，包括肾病和 免疫抑制患者中的出血性膀胱炎。关于为什么BKV是热带的 肾脏以及它的取向是否仅限于泌尿系统。此外，制约经济增长的因素 BKV产生性感染和持续性感染之间的平衡尚不清楚。 此应用程序侧重于了解导致高效或持久的细胞响应的基础 感染。BKV在原代人肾近端小管上皮细胞(RPTE)中经历生产性感染， 以广泛的细胞死亡和大约40个感染粒子/细胞的释放为特征。相比之下，BKV 在血管内皮细胞(VEC)中建立低水平的持续感染，导致最小的细胞病变 在两个月的传代过程中，大约10%的细胞表达病毒蛋白。RNA-Seq显示 BKV在RPTE和RPTE中诱导许多受E2F家族转录因子调控的细胞周期基因 VEC。此外，许多干扰素刺激基因(ISGs)在感染BKV后上调 VEC，而不是RPTE。 这项研究将使用人类原代血管内皮细胞培养来确定影响感染的因素 结果：富有成效或持之以恒。单细胞转录将应用于模拟和BKV感染的血管内皮细胞 评估细胞亚群对感染的反应，并确定起限制作用的基因 BKV复制。BKV感染在血管内皮细胞中对干扰素反应的激活将使用两种方法进行评估 分子和功能(CRISPR基因敲除)方法，以确定途径的组件 对限制病毒感染至关重要。特定的病毒突变将被用来确定病毒的功能是什么 诱导干扰素反应所需的，以及在病毒感染的哪个阶段 诱导性。点击化学将被用来确定哪些细胞因子调节 多产且持续的感染。将使用多种方法的组合来识别细胞基因 区分限制性和生产性感染细胞。 这项工作将显著提高我们对BKV致病机理的了解，通过表征病毒的相互作用 通过识别促进或限制BKV感染的细胞因子 并将为抗病毒药物的设计提供有益的见解。

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.