PROJECT #2: MECHANISMS CONTROLLING NEUROINVASION OF BRAIN CELLS BY JCPYV

项目概要-项目2 JCPyV与其宿主的初始相互作用涉及识别细胞上的特异性受体复合物。 这些受体的识别导致病毒穿透宿主细胞膜，将病毒粒子运输到宿主细胞膜。 内质网，并最终将dsDNA基因组传递到细胞核。我们的团队， 在该计划项目的支持下，阐明了JCPyV受体复合物的关键组分， 确定主要衣壳蛋白VP 1负责将病毒引导至ER。我们开发 新的工具，不仅研究实验室适应株的JCPyV，但也突变形式的病毒， 在PML患者的大脑中出现。这些突变形式的JCPyV已经失去了 识别唾液酸受体，因此在大多数检查的细胞类型中不再具有感染性。 我们假设这些突变体要么识别尚待鉴定的替代受体， 它们已经获得了绕过细胞表面直接在细胞间传播的能力 受体。我们在项目2中的方法是使用在核心B中开发的假病毒来进一步探索潜力 这些突变体的受体利用率。我们还将探索这些突变体的可能性， 通过将突变工程化到感染性JCPyV克隆中并在感染后生长， 将基因组转染到生长为汇合单层的不同细胞类型中。无论 感染机制（受体介导或直接细胞间传播）这些病毒必须全部运输到ER 从而开始脱膜过程，最终将它们的基因组运送到细胞核。在上一个供资周期 我们发现二氢喹唑啉酮化合物Retro-2cycl有效地阻止JCPyV运输到 ER和大大减少初始感染和感染传播。我们现在的目标是确定它的机制， 作用，以确定其细胞靶点，并优化现有的化合物，使治疗窗口 可以确定抑制。

PROJECT SUMMARY - PROJECT 2 The initial interaction of JCPyV with its host involves recognition of specific receptor complexes on cells. Recognition of these receptors leads to virus penetration of the host cell membrane, trafficking of the virion to the endoplasmic reticulum, and the eventual delivery of the dsDNA genome to the cell nucleus. Our team, supported by this program project, elucidated the critical components of the JCPyV receptor complex and determined that the major capsid protein VP1 was responsible for directing the virus to the ER. We developed novel tools to study not only lab adapted strains of JCPyV but also mutant forms of the virus that have been reported to arise in the brains of patients with PML. These mutant forms of JCPyV have lost the ability to recognize the sialic acid receptor and as a consequence are no longer infectious in most cell types examined. We hypothesize that these mutants either recognize alternative receptors which are yet to be identified or that they have gained the capacity to spread directly from cell to cell bypassing the requirement for cell surface receptors. Our approach in project 2 is to use pseudoviruses developed in core B to further explore potential receptor usage by these mutants. We will also explore the possibility that these mutants, are capable of direct cell to cell spread by engineering the mutations into an infectious JCPyV clone and following their growth after transfection of the genomes into different cell types grown as confluent monolayers. Regardless of the mechanism of infection (receptor mediated OR direct cell to cell spread) these viruses must all traffic to the ER to begin the uncoating process for eventual delivery of their genomes to the nucleus. In the last funding cycle we discovered that the dihydroquinozolinone compound Retro-2cycl potently prevents JCPyV trafficking to the ER and substantially reduces initial infection and infectious spread. Our goal now is to define its mechanism of action, to identify its cellular targets, and to optimize the existing compound so that a therapeutic window of inhibition can be determined.