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

项目摘要--项目2 JCPyV与宿主的最初相互作用涉及识别细胞上的特定受体复合体。 对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 内质网，以及dsDNA基因组最终传递到细胞核。我们的团队， 在该项目的支持下，阐明了JCPyV受体复合体的关键成分和 确定主要衣壳蛋白VP1负责将病毒引导到ER。我们开发了 新的工具不仅可以研究实验室适应的JCPyV毒株，还可以研究已经被 据报道，它出现在PML患者的大脑中。这些JCPyV的突变形式已经失去了 识别唾液酸受体，因此在所检测的大多数细胞类型中不再具有传染性。 我们假设，这些突变体要么识别尚未确定的替代受体，要么 它们已经获得了在细胞之间直接传播的能力，绕过了对细胞表面的要求 感受器。我们在项目2中的方法是使用在核心B中开发的伪病毒来进一步探索潜力 这些突变体对受体的使用。我们还将探索这些突变体是否能够直接 通过将突变改造成具有感染性的JCPyV克隆并跟随它们的生长 将基因组转入以融合单层形式生长的不同类型的细胞。不管是什么 感染机制(受体介导或直接细胞间传播)这些病毒必须全部传播到急诊室 以开始最终将它们的基因组输送到细胞核的揭开涂层的过程。在上一个资金周期中 我们发现，二氢喹唑酮化合物Retro-2 Cycl1有效地阻止了JCPyV向 并大大减少了初始感染和传染病的传播。我们现在的目标是定义它的机制 作用，以确定其细胞靶点，并优化现有的化合物，以使治疗窗口 可以确定抑制率。

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.